Chemicals
5 articles
Chemical Exposure Levels
Six abbreviations are used to describe the administered doses reported in the literature. These terms indicate whether the dose caused death (LD) or other toxic effects (TD) and if the dose was administered as a lethal concentration (LC) or toxic concentration (TC) in the inhaled air. In general, the term "Lo" is used where the number of subjects studied was not a significant number of the population or the calculated percentage of subjects showing an effect was listed as 100. These are the used definition: TDLo, Toxic Dose Low: The lowest dose of a substance introduced by any route, other than inhalation, over any given period of time and reported to produce any toxic effect in humans or to produce carcinogenic, neoplastigenic, or teratogenic effects in animals or humans. LDLo, Lethal Dose Low: The lowest dose (other than LDS0) of a substance introduced by any route, other than inhalation, over any given period of time in one or more divided portions and reported to have caused death in human or animals. LD50, Lethal Dose Fifty: A calculated dose of a substance which is expected to cause the death of 50% of an entire defined experimental animal population. It is determined from the exposure to the substance by any route other than inhalation of a significant number from that population. Other lethal dose percentages, such as LDI, LDi0, LD30, and LD99, may be published in the scientific literature for the specific purposes of the author. Such data would be published in the Registry if these figures, in the absence of a calculated lethal dose (LDS0), were the lowest found in the literature. TCLo, Toxic Concentration Low: The lowest concentration of a substance in air to which humans or animals have been exposed for any given period of time that has produced any toxic effect in humans or produced a carcinogenic, neoplastigenic, or teratogenic effect in animals or humans. LCLo, Lethal Concentration Low: The lowest concentration of a substance in air, other than LCS0, which has been reported to have caused death in humans or animals. The reported concentrations may be entered for periods of exposure which are less than 24 hours (acute) or greater than 24 hours (subacute and chronic). LC50, Lethal Concentration Fifty: A calculated concentration of a substance in air, exposure to which for a specified length of time is expected to cause the death of 50% of an entire defined experimental animal population. It is determined from the exposure to the substance of a significant number from that population.
Conductive Adhesives Comparison Table
Product Form Service Temp. Cure Temp . Conductive Media (%) Carrier Binder Solvent Conductive Graphite, 50g paint 140°C graphite 22% gel water Conductive Graphite, 30g paint binder 93°C service 204°C graphite 20% cellulose resin Isopropanol Leit‐C‐Plast 15g putty 120°C carbon proprietary putty ethanol Colloidal Silver, 25g or 100g paint 200°C silver 60% lacquer Methyl Ethyl Ketone High Performance Silver Paste, 50g paste cryogenic to 927°C 2Hr/RT 2Hr/93°C silver 60% inorganic silicate water High Performance Ceramic ADH, 1PT, 50g paste 1650°C 2Hr/RT 2Hr/93°C >60% Al2O3 inorganic silicate water High Temperature Carbon Paste, 50g paste 2000°C 2Hr/RT 2Hr/93°C 50 ‐ 60% carbon inorganic silicate water High Performance Nickel ADH, 1 PT, 50g paste 538°C 2Hr/RT 2Hr/93°C >70% Nickel inorganic silicate water Continued... Product Mech. Strength Sheet Res. @1mil (ohms/sq ) RoHs Compliant Application Thermo‐ conductivity (BTU‐in/ft2‐ hr‐F°) Mounting High Vacuum EDX/ EDS EMI/RF Shield Cleaving Conductive Graphite, 50g low 30 X X X X Conductive Graphite, 30g low 1200 X X X X Leit‐C‐Plast 15g medium X X X X Colloidal Silver, 25g or 100g medium 0.02‐0.04 X X High Performance Silver Paste, 50g medium 0.08 X X 63.2 High Performance Ceramic ADH, 1PT, 50g low insulator X <6 High Temperature Carbon Paste, 50g low 4.6 X X 6.9 High Performance Nickel ADH, 1 PT, 50g low 2.0 X X 18
Hydrogenated Nitrile Butadiene Rubber (HNBR)
Properties and Applications HNBR is widely known for its physical strength and retention of properties after long‐term exposure to heat, oil, and chemicals. Depending on filler selection and loading, HNBR compounds typically have tensile strengths of 20‐31MPa when measured at 23°C. Compounding techniques allow for HNBR to be used over a broad temperature range, ‐40° to 165°C, with minimal degradation over long periods of time. For low‐temperature performance, low ACN grades should be used; high‐temperature performance can be obtained by using highly saturated HNBR grades with white fillers. As a group, HNBR elastomers have excellent resistance to common automotive fluids (e.g., engine oil, coolant, fuel, etc.) and many industrial chemicals. Like NBR, fluid and chemical resistance improves as the ACN content is increased. The unique properties attributed to HNBR have resulted in wide adoption of HNBR in automotive, industrial, and assorted, performance‐demanding applications. On a volume basis, the automotive market is the largest consumer, using HNBR for a host of dynamic and static seals, hoses, and belts. HNBR has also been widely employed in industrial sealing for oil field exploration and processing, as well as rolls for steel and paper mills. Chemistry and Manufacturing Process The basic structure of an HNBR elastomer is provided in Figure 1. As outlined below, the process begins with the production of an emulsion‐polymerised NBR. This polymer is then dissolved in an appropriate solvent. After the dissolution process is complete, the addition of hydrogen gas, in conjunction with a precious metal catalyst at a designated temperature and pressure, brings about a selective hydrogenation to produce a "highly saturated nitrile" (HSN) polymer. Even today, HNBR is still sometimes referred to as "HSN". Basic Recipes There are a wide variety of acrylonitrile (ACN) content polymers available in the HNBR market today. They range from approximately 17 to 50% ACN. The ACN content not only controls fluid resistance but also impacts the low temperature performance. If the ACN content of the polymer is increased, the volume swell of the associated compound will decrease while the low‐temperature flexibility will become poorer. Alternatively, if one decreases the ACN level of the polymer, the associated compound will have higher volume swell and improved low temperature flexibility. Likewise, as the hydrogenation level is increased, the heat and ozone resistance improves but the dynamic hysteresis increases. If you decrease the hydrogenation level, the heat and ozone resistance is not as good but the dynamic hysteresis improves significantly. The other characteristic imparted by the hydrogenation level is the type or selection of the appropriate cure system. Lastly, the wide range of Mooney viscosities available permits the compounder to choose a product which best suits their specific method of manufacturing (e.g., compression, transfer, or injection moulding vs. extrusion). Today, HNBRs range in Mooney viscosity from 50 to approximately 150 when measured at ML(1+4) @100°C. A typical HNBR recipe is listed in the table below. Ingredients Phr Polymer Filler 100 Filler 40 ‐ 100 Plasticizer 0 ‐ 20 Metal Oxide 0 ‐ 5 Anti-degradents 1.5 ‐ 3 Process Aids 0 ‐ 3 Coagent 0 ‐ 10 Curative 5 ‐ 12 Processing and Vulcanisation Mixing All of the commercially available HNBRs can be mixed either on a two‐roll mill or with an internal mixer. Most HNBRs are mixed by the use of internal mixing equipment. This is done to improve the quality of the finished compound while also significantly reducing the typical mix times incurred when done on a two‐roll mill. Typically, HNBRs are two‐pass mixed in an internal mixer where the first pass through the mixer withholds the cure chemicals and is typically dropped out of the mixer at approximately 140°C. This masterbatch is then run through the mixer a second time adding the cure chemicals and again dropped from the mixer at approximately 100‐110°C. Milling The milling of an HNBR compound is typically quite easy. HNBR compounds tend to build heat quickly; therefore, the use of full cooling capabilities is typically recommended. Normally one should start by setting the mill gap at approximately ¼". This ensures that you will get the shear action required to finish the mixing process while being thin enough to dissipate any excess heat generated during this process. One would normally drop the compound on the mill from an internal mixer from above or feed a stored compound one sheet at a time near the ends of the mill. After banding the compound on the mill, cross‐cutting the compound 5 ‐ 7 times from each end is usually adequate to complete this process. Batching off of the mill can easily be accomplished via automatic systems or by hand. Vulcanisation HNBR elastomers are typically cured with either peroxide or sulphur/sulphur‐donor cure systems. Laboratory comparisons of sulphur/sulphur‐donor and peroxide cured HNBR compounds indicate that peroxide curing provides better compression set and heat resistance. Because HNBR has fewer highly reactive allyl position hydrogens versus other diene‐based elastomers, such as NBR and SBR, it is necessary to add 50‐100% more peroxide in order to produce excellent curing characteristics. Many kinds of peroxides are available for curing HNBR. However, it is important to select one that is the most suitable based on the process and cure temperature that will be utilised to produce the finished parts. Since peroxides have different molecular weights and decomposition temperatures, it is imperative to select the correct one based on the criteria noted above or one can greatly affect the processability and cost‐effectiveness of producing the finished goods in question. As in all peroxide cured material, vulcanisation in the presence of oxygen causes reversion and thus leaves a sticky surface on the cured part. Therefore, one must take care when using pressureless cures and purge the autoclave prior to pressuring up for curing. When compared to SBR or NBR, the curing speed tends to be slower; therefore, to increase the curing speed a secondary accelerator should be employed in combination with the primary accelerator. Long curing times are required when thiazole based (MBTS) or sulfenamide‐based (CBS, OBTS) primary accelerators are used. To speed up the curing process, a small quantity of guanidine‐based (DPG) or thiuram‐based (TMTM) secondary accelerator is used in combination with the primary accelerator. Even when using a thiuram‐based (TMTD, TETD) primary accelerator, the addition of a thiazolebased (MBT) secondary accelerator will shorten the time required for curing. The use of dithiocarbamate (ZEDC) as the primary accelerator is undesirable since the scorch time will be shortened. Producers The hydrogenation of various diene‐containing elastomers has been well known for many years. Berthelot began the earliest experiments in 1869, with many other workers carrying out numerous experiments under different conditions and with different catalyst systems. Pummerer et al., in the early 1920s and Staudinger about 1930 were notable contributors to the hydrogenation of polymer systems. Hydrogenated nitrile butadiene rubber or HNBR was first developed in the late '70s and early '80s. Though commercial production did not begin until 1984, there were numerous companies looking at the feasibility of producing this type of elastomer. Of those who chose to evaluate this polymer, three companies emerged: Bayer Corporation, Zeon Corporation, and Polysar. Of these three, Zeon Corporation was the first to commercialise HNBR in March of 1984 with Bayer and Polysar very close behind. Zeon Corporation's initial manufacturing site was in Takaoka, Japan. The other initial manufacturing site was Polysar's in Orange, Texas. However, Polysar eventually sold that business to Bayer Corporation (now Lanxess) who now owns and operates that facility. Conclusion HNBR has been the fastest growing specialty elastomer in the last decade. With its excellent cost‐performance balance for the most demanding of applications; HNBR is the ideal choice for applications needing excellent physical properties, as well as oil, heat, and/or chemical resistance. Source: http://www.iisrp.com/WebPolymers/AboutRubber/03HNBR16Aug2012.pdf 09/09/2013
Spherotech Microparticles Technical Notes
Download Technical Data Sheets (further detail below): STN-1: Particles Coating Procedures STN-2: Determination of Antibody Binding to Particles STN-3: Binding Capacity of Avidin Magnetic Particles STN-4: Binding Capacity of Gt-anti-Ms-IgG Magnetic Particles STN-5: Binding Capacity of Streptavidin Magnetic Particles STN-6: Binding Capacity of Biotin Magnetic Particles STN-7: Separation of Mononuclear Cells from Peripheral Blood using SPHERO Gt-anti-Ms-IgG Magnetic Particles STN-8: Calibration and Performance Tracking of Flow Cytometer Using SPHERO Calibration Particles STN-9: Measuring MEF with Flow Cytometer Using SPHERO Rainbow Calibration Particles STN-10: Magnetic Particles Enzyme Immunoassay (MPEIA) using UltraMag Separator System (UMS-4000) STN-11: Magnetic Particles Coated with Pepsin, Papain and Trypsin STN-12: Protein A Coated Magnetic Particles STN-14: Determining PMT Linearity in Flow Cytometers Using the SPHERO PMT Quality Control Excel Template STN-15: Measuring Absolute Cell Count Using SPHERO ACCUCOUNT Fluorescent Particles STN-16: Covalent Coupling of Proteins to Microbeads Using a Heterobifunctional Coupling Agent STN-17: Determination of a Flow Cytomerter’s Sensitivity Using Detection Efficiency (Q) and the Background Light Level (B) STN-18: Introduction to an Easy-To-Use Technique For The Setting of Flow Cytometer Compensation Using COMPtrol Antibody Capture Beads as a Substitute For Cells STN-19: Covalent Coupling of T25 DNA to Carboxyl Magnetic Particles STN-20: Activation Techniques for Hydroxyl Magnetic Particles STN-21: Streptavidin Particles Uses and Protocols STN-22: Goat anti-Mouse Particle Uses and Protocols STN-23: Magnetic Antibody Coated Particles for Cell Isolation STN-1: Particles Coating Procedures Currently, there are several methods of attaching biological ligands to polystyrene particles. These methods include adsorption to plain polystyrene particles, covalent attachment to surface functionalised particles, and attachment of the ligand of interest to particles that are pre-coated with a binding protein such as Streptavidin, Protein A or Protein G. Presented in this Spherotech Technical Note are protocols such as adsorption, covalent coupling, and other methods used to attach ligands to polystyrene particles. Procedures and Discussion: The following information explains generalised protocols for the attachment of ligands to polystyrene particles. These protocols are easily optimised to meet the requirements of specific applications. The following protocols are developed by Spherotech for the convenience of SPHERO™ microparticle users. They are to be utilised only as initial conditions. Spherotech encourages the optimisation of the coating conditions by changing the buffer, pH or reagents concentration. In general, polyclonal antibodies are coated to polystyrene particles by adsorption without using any coupling agents. The binding of polyclonal antibodies to polystyrene particles is strong. However, care should be taken not to overload the antibodies to the particles. If overloading occurs, leaching of the coated antibody will happen during storage. This is due to the weak interaction between antibody molecules compared to the interaction of antibody molecules on the surface of polystyrene particles. SpheroTechnical information: Particle coating using passive adsorption Amino particles with ligands or proteins using EDC and covalent coupling Carboxyl particles with avidin or other proteins using EDC covalent coupling Covalent coupling using a two-step EDC coupling protocol Avidin particles with biotinylated proteins using affinity coupling Covalent coupling of protein to hydroxyl particles using cyanogen bromide (CNBr) Dimethylamino particles with DNA using ionic interaction coupling Ligands to modified amino proteins covalent coupling using SPDP Periodate oxidation of polysaccharides and coupling to amino particles Carboxyl polystyrene particles with amino-modified oligonucleotides STN-2: Determination of Antibody Binding to Particles The amount of the antibody binding to the particles after coating can be determined by using the subtraction method as follows: Measure the absorbance of the antibody solution to be used for coating at 280nm (OD280). The IgG has OD280 of ~1.4/mg. Pellet the particles to be coated by centrifugation or magnetic separation. STN-3: Binding Capacity of Avidin Magnetic Particles Materials: Avidin magnetic particles, 1% w/v, QVM-40-10 , Lot No. J01, 4.35μm Biotin-FA conjugate, Lot No. 021688, 533 nM in 1% diluent. (IBS containing 1% normal goat serum and 1% foetal bovine serum ) Procedure: Add 50, 100,150, 200, 300 & 400 μl of Avidin magnetic particles to six 12 x 75mm test tubes. Add 1mL of Biotin-FA conjugate to each tube. Vortex and incubate at ambient temperature for at least 30 minutes with occasional shaking. Adjust the fluorimeter for excitation and emission at 490 and 520nm respectively STN-4: Binding Capacity of Gt-anti-Ms-IgG Magnetic Particles Materials: Gt. anti-Ms IgG magnetic particles, 1% w/v, QMM-40-10 , Lot No. J01, 4.35μm Ms-IgG FITC, Lot No. 10952, 5μg/mL in 1% diluent ( IBS containing 1% normal goat serum and 1% foetal bovine serum) Procedure: Adjust fluorimeter for excitation and emission at 490 and 520nm respectively. Set 100% emission with the Ms IgG-FITC solution. Vortex the Goat anti-Ms IgG(H&L) magnetic particles and add 50, 100, 150, 200,300, and 400μL into six 12 x 75mm tubes. Pellet the particles and aspirate the supernatant. Add 1 ml of Ms IgG-FITC solution to every tube containing the pelleted particles. Vortex and incubate for 30 minutes. Separate the Goat anti-Ms IgG(H&L) magnetic particles from the conjugate and read the fluorescence of the supernatant. STN-5: Binding Capacity of Streptavidin Magnetic Particles Materials: Streptavidin magnetic particles, 1% w/v, QSVM-40-5 Lot No. Y01, 4.35µm Biotin-FA solution, Spherotech, Lot No. 021688, 533 nM in 1% diluent (IBS containing 1% normal goat serum and 1% foetal bovine serum) Procedure: Adjust the fluorimeter for excitation and emission at 490 and 520nm respectively. Set 100% emission with the Biotin-FA solution. Add 50, 100, 150, 200, 300, and 400µL of Streptavidin magnetic particles to six 1.5ml microfuge tubes. STN-6: Binding Capacity of Biotin Magnetic Particles Materials: Biotin magnetic particles, 1% w/v, QTM-40-10 Lot No. J01, 4.32µm Avidin-FITC solution, Jackson ImmunoResearch, #003-090-083, Lot No. 21452, 5µg/mL in 1% diluent.(IBS containing 1% normal goat serum and 1% fetal bovine serum) Procedure: Adjust the fluorimeter for excitation and emission at 490 and 520 nm, respectively. Set 100% emission with the Avidin-FITC solution. Add 25, 50, 75, 100, and 200µL of Biotin magnetic particles to five 1.5mL microfuge tubes. Separate the particles magnetically and remove the supernatant. Add 1 mL of Avidin-FITC solution to each tube, vortex and rotate at room temperature for one hour. STN-7: Separation of Mononuclear Cells from Peripheral Blood using SPHERO™ Gt-anti-Ms-IgG Magnetic Particles Materials: Gt anti-Ms IgG Magnetic Particles, 1% w/v, QMM-40-10 or QMMXA-40-10 , ~2e8 particles/mL Appropriate monoclonal antibody (anti-CD3, CD4 or CD8, etc.) Appropriate FITC conjugates Dulbecco PBS Fetal bovine serum Histopaque Paraformaldehyde fixative FlexiMag Separator, Jr., QFMJ-1000 Procedure: Collect peripheral blood by venipuncture of the antecubital vein. Draw blood into heparin Vacutainer tubes, transfer to 50mL polypropylene centrifuge tubes, and dilute with an equal volume of calcium and magnesium-free Dulbecco’s phosphate buffered saline. Layer 10 to 20mL aliquots of diluted blood onto an equal volume of Histopaque in 50mL tubes. Centrifuge for 30 minutes at ambient temperature using a centrifugation force of 400g at the blood/Histopaque interface. Aspirate the lymphocyte band into a 15mL centrifuge tube and bring the volume to 14mL with Dulbecco PBS containing 2% v/v heat-inactivated fetal bovine serum or 5% plasma protein fraction. Pellet the cells by centrifugation at 4°C for 7 minutes at 450g, wash with 14mL of PBS, and recentrifuge. Resuspend the final cell pellet in 1-2mL buffer, and a viable cell count is performed. Only cell preparations with a viability of >95% should be used. SpheroTechnical information: Separation of Mononuclear Cells From Peripheral Blood Coating of Anti-mouse Igg Magnetic Particles With Monoclonal Antibody Incubation of Magnetic Particles and Cells Detection of Cell Depletion by Immunofluorescence STN-8: Calibration and Performance Tracking of Flow Cytometer Using SPHERO™ Calibration Particles The SPHERO™ Calibration Particles are versatile, stable, economical and convenient to use. These particles contain a mixture of fluorochromes which are spectrally similar to many of the fluorochromes used in flow cytometry. As a result, they are used for routine alignment, day-to-day performance verification, and long-term performance tracking of several channels of flow cytometers in one run. These particles are very stable since the fluorochromes are entrapped inside the particles instead of being located on the surface. They are packaged in a convenient dropper bottle to facilitate dispensing and storage. The diluted particles can be stored for later use if desired to reduce costs. These products and their uses are described briefly as follows: Rainbow Calibration Particles (RCPs): The RCPs are designed for the routine calibration of most available channels in any flow cytometer. For example, these particles are used to verify the instrument setup and to check the linearity and sensitivity of the instrument. If factory-recommended procedures are used for instrument setup, we recommend that the RCPs be included in QC programs to track the long-term and day-to-day performance. SpheroTechnical information: Rainbow Calibration Particles (RCPs) for creating calibration curves and determining sensitivity Ultra Rainbow Calibration Particles (URCPs) for creating calibration curves and determining sensitivity Rainbow Fluorescent Particles (RFPs) for checking system setting and alignment Ultra Rainbow Fluorescent Particles (URFPs) for checking system setting and alignment Yellow Calibration Particles (YCPs) for the calibration of the FL1 channel Allophycocyanin Calibration Particles (ACPs) for calibrating the Allophycocyanin channel Blank Calibration Particles (BCPs) for setting the fluorescence threshold Recommended Flow Cytometry QA Procedures including the following: Daily Alignment , Setting the Threshold, Verification of Setting and Validation of Logarithmic Amplifier Linearity and Sensitivity, Compensation & Creating Levey-Jennings Charts STN-9: Measuring MEF with Flow Cytometer Using SPHERO™ Rainbow Calibration Particles The Molecules of Equivalent Fluorochrome (MEF) value is useful for flow cytometer users. However, the Quantum Yield of most fluorochromes usually changes upon binding to the cells. As a result, the actual number of fluorochrome molecules binding to the cells may be different than the expected MEF value. In addition, the MEF value will vary depending on the measurement method. There are several methods for measuring the MEF value. Most commonly, a standard curve is generated with a fluorimeter using solutions of fluorochrome in various concentrations. The fluorescence of these solutions is then compared to the intensity of labeled cells or particles less the fluorescence of a blank particle or non-labeled cell. This provides the equivalent concentration of fluorochrome. The MEF value is then calculated by dividing the equivalent fluorochrome concentration by the number of cells or particles used. SpheroTechnical information: Methods for measuring the MEF value Measuring relative MEF values using the Ultra Rainbow Calibration Particles (URCPs) and Rainbow Calibration Particles (RCPs) Normalising different instruments using URCPs or RCPs Quantitative flow cytometry using URCPs or RCPs STN-10: Magnetic Particles Enzyme Immunoassay (MPEIA) using UltraMag Separator System (UMS-4000) Magnetic Particle Enzyme Immunoassay (MPEIA) is an immunoassay method for the isolation of antibody/antigen complexes on a solid phase surface of magnetic microparticles. MPEIA has been used to automate the measurement of large molecules such as markers associated with cardiac, fertility, cancer, metabolic, hepatitis, and thyroid testing. Procedure: In the wells of a microEIA plate, make serial 2-fold dilutions of rabbit, human, or mouse IgG in 1% bovine serum albumin (BSA), adding 100µL /well. The control wells should receive 100µL of 1% BSA without IgG. Add 50µL of a 0.25% (w/v) suspension of Spherotech QCM-40-10 magnetic particles coated with antibodies to rabbit, human or mouse IgG. Dilute the particles in a buffer consisting of 1% casein hydrolysate and 0.05% Tween 20 in phosphate buffer saline, pH 7.2. SpheroTechnical information: A protocol for the development of a Magnetic Particle Enzyme Immunoassay (MPEIA) The advantages of MPEIA over coated well EIA Selected references showing use of magnetic particles in immunoassays STN-11: Magnetic Particles Coated with Pepsin, Papain and Trypsin Preparation of IgG F(ab’)2 Fraction Using Pepsin Coated Magnetic Particles (QPEPM-40-2) The QPEPM-40 consists of 4µm magnetic particles covalently linked to pepsin from porcine stomach mucosa. These particles efficiently cleave IgG from various species producing F(ab’)2 and other lower molecular weight products within a few hours. After digestion, Pepsin-Magnetic particles can be easily removed magnetically from the reaction vessel, leaving no pepsin in the solution. As a result, the cleavage of IgG can be effectively controlled. The supernatant containing products from the cleavage and any residual IgG can be further purified chromatographically or by using Protein A magnetic particles ( QPAMX-10 ). Preparation of IgG Fab Fraction Using Papain Coated Magnetic Particles (QPAPM-40-2) The PAPM-40 consists of magnetic particles (4µm) covalently linked to Papain from Papaya latex. These particles efficiently cleave IgG into Fab and Fc fragments within a few hours. After digestion, Papain-Magnetic particles can be easily removed from the reaction vessel magnetically, leaving no Papain in the solution. As a result, effectively stops further cleavage of IgG. The supernatant containing products of cleavage and any undigested IgG can be further purified chromatographically. This procedure allows the controlled preparation of essentially pure Fab in simple two steps. In addition, papain magnetic particles can be reused repeatedly without a significant loss in activity. SpheroTechnical information: A procedure for the controlled preparation of essentially pure F(ab’)2 using Pepsin Magnetic Particles A procedure for the controlled preparation of essentially pure Fab using Papain Coated Magnetic Particles The advantages of using enzymes covalently linked to magnetic particles over enzymes attached to nonmagnetic gel, latex, or soluble enzymes Selected references using Magnetic Particles Coated with Pepsin and Papain STN-12: Protein A Coated Magnetic Particles Protein A is a 42kD polypeptide isolated from Staphylococcus aureus which has a specific binding affinity for the Fc region of IgG from several species. Each Protein A molecule has four IgG binding sites. In addition, protein A binds to IgG without interfering with the antigen-binding site of the immunoglobulin. The Protein A-coated magnetic particles provide a quick, easy, and economical way for the capture of antigen-specific antibodies used in the purification of recombinant antigens. In the past, protein A linked gel matrix has been routinely used for isolating IgG from human, mouse, and rabbit serum. However, protein A covalently bound to magnetic particles increases the reaction kinetics while reducing the capture time of antigen-specific antibodies. As a result, Protein A-coated magnetic particles are uniquely suited for isolating IgG from limited volume samples without dilution or loss. They can also be used to capture and concentrate low-level IgG in large-volume samples. In addition, Protein A-coated magnetic particles can be repeatedly used without a significant loss in their ability to bind IgG. SpheroTechnical information: The affinity of Protein A coated beads to IgG from different species Product details for the various Protein A linked magnetic particles offered from Spherotech A protocol for the purification of IgG from hybridoma tissue culture for clone selection A protocol for the isolation of specific cells from blood (B, T and HLA) using Protein A Magnetic Particles A protocol for an All-Purpose “Fish-Hook” for isolating specific antigen from a mixture A protocol for the reuse of Protein A Linked Magnetic Particles STN-14: Determining PMT Linearity in Flow Cytometers Using the SPHERO™ PMT Quality Control Excel Template The fluorescence linearity of flow cytometers is affected by optical alignment, laser power, electronic offsets, and amplifier calibration1. In addition, it is important to monitor and validate flow cytometers’ performance due to the nature of the information obtained during diagnostic testing. As a result, it is recommended that the linearity of the flow cytometer is determined on a monthly basis after instrument repair and after instrument relocation (2). The SPHERO™ Calibration Particles and SPHERO™ PMT Quality Control Excel Template (PMT QC Template) are designed for linearity calibration and long-term performance tracking of flow cytometers. They will help flow cytometer users verify the operation of their instruments. The PMT QC Template is a valuable tool for determining the linearity of log amplifies. The information acquired from this template should be implemented into flow cytometer calibration documentation. The user can determine a schedule for routine maintenance procedures and tolerance limits of linearity based on instrument trends or malfunctions using this template. The SPHEROTM Rainbow Calibration Particles (RCPs) and Ultra Rainbow Calibration Particles (URCPs) contain a mixture of similar size particles with different fluorescence intensities. SpheroTechnical information: The use of the Rainbow Calibration Particles (RCPs) for the determination of the logarithmic amplifier linearity The use of the PMT QC Template to determine the number of related fluorophores for an unknown sample or other particles References using the RCPs for the standardisation of flow cytometers STN-15: Measuring Absolute Cell Count Using SPHERO™ ACCUCOUNT Fluorescent Particles Assays for cell counting using flow cytometry and calibrated fluorescent particles are rapid and accurate. The single platform method that enumerates T-cells by counting the identifier cells in either a precise known cell volume or an internal ‘spike’ of a known number of calibrated fluorescent particles by flow cytometry is simple and efficient (1). These assays allow the counting of T-cells during anti-T-cell globulin treatment of cardiac, lung, and renal transplant patients (2). In addition, laboratories can determine the absolute count of CD4 and CD8 T-cells to estimate HIV disease progression with the single platform method (3). Calibrated fluorescent particles and flow cytometry are also used to count platelets in a wide range of murine models of platelet disorders (4). It is also possible to count other various cell types with flow cytometry and calibrated fluorescent particles. SpheroTechnical information: A description of using Spherotech AccuCount beads and flow cytometry for the ratio-metric method of absolute counting A protocol for using the Spherotech AccuCount beads for cell enumeration Calculation necessary for using absolution counting beads for cell enumeration References using the AccuCount beads STN-16: Covalent Coupling of Proteins to Microbeads Using a Heterobifunctional Coupling Agent Covalent coupling of lysozyme to amino-polystyrene particles utilising a heterobifunctional coupling agent, SPDP, and to aldehyde particles was studied using Particle Concentration Fluorescence Immunoassay (PCFIA). The results showed that these methods can be used to couple lysozyme covalently to amino-polystyrene and aldehyde particles. The modification of amino-polystyrene particles with SPDP offers the advantage that the resulting pyridyl disulfide particles can be used to react specifically with the sulfhydryl group of proteins, or they can be reduced further with DTT to form sulfhydryl particles. The aldehyde particles can be used to couple covalently to proteins without any coupling agent. The activity of the lysozyme-coated particles depends upon the orientation of lysozyme on the particles. Higher activity was obtained when the amino groups of the lysozyme were utilised to couple to the particles covalently. SpheroTechnical information: A protocol for covalently coupling lysozymes to amino-polystyrene particles through either EDC coupling on SPDP modification A protocol for the covalent coupling of lysozymes to aldehyde particles without the use of a coupling agent How the activity of coated particles varies with the coupling methods used and depends on the orientation of lysozyme on the surface of the microparticles Various coupling strategies, including passive adsorption on particles with different surface charges and covalent coupling using different functionalised particles and coupling agents, can be used to optimise the activity of immobilised protein on microparticles STN-17: Determination of a Flow Cytomerter’s Sensitivity Using Detection Efficiency (Q) and the Background Light Level (B) In order to obtain accurate and scientifically sound flow cytometric data it is critical to use standardisation techniques and a robust and reliable instrument. The standardisation protocol should address specific operational parameters to determine the performance of the instrument at any point in time. The flow cytometer's optic, fluidic, and electronics design should be simplistic while still being sensitive and effective. Experimental Procedure: One major parameter to be included in the standardisation protocol is a test for sensitivity. Sensitivity is an important parameter since it defines the ability to detect particles above the background. However, sensitivity determination should also include resolution, the ability to resolve dim particles. This can be measured using Spherotech Rainbow Calibration Particles with eight intensities, QRCP-30-5A (Rainbow Calibration Particles, 8 peaks, 1E7/mL, 3.0-3.4um, 5mL). Since each intensity of the QRCP-30-5A has been calibrated to the molecules of equivalent fluorophores (MEF), it can be used to quantify sensitivity. Figure 1 is the histogram of the QRCP-30-5A on Stratedigm's S1000 cutting-edge flow cytometer in the PE channel. See http://statedigm.com/instrumentation for more information on Stratedigm flow cytometers. A flow cytometer's sensitivity can be described as the detection efficiency (Q) and the background light level (B). The detection efficiency is how well light is collected in the cytometer, while the background light level shows how much noise is created by the instrument in the background. In order to determine the efficiency (Q) and the background light level (B), the MEF values of blank beads and another dim population must be known. These values can be obtained using the Spherotech PMT QC Template. SpheroTechnical information: How the Spherotech 8 peak Rainbow Calibration Particles ( QRCP-30-5A ) provides robust and reliable data when a standardisation protocol is successfully adopted and implemented An introduction to using the Rainbow Calibration Beadsfor the calculation of a flow cytometer's sensitivity by measuring the Detection Efficiency (Q) and the Background Light Level (B) Why a flow cytometer standardisation protocol should include the statistical analyses of sensitivity measurements to promote innovative, scientifically sound experimental results STN-18: Introduction to an Easy-To-Use Technique For The Setting of Flow Cytometer Compensation Using COMPtrol Antibody Capture Beads as a Substitute For Cells When setting up multicolour flow cytometry experiments proper compensation is extremely important since these experiments provide complex data. Compensation helps correct spectral overlap to match the various fluorophores used during cell staining, after which the data becomes easier to interpret. Compensation using cells for single-colour staining provides autofluorescence levels that are the same as those obtained during multicolour staining and are independent of the antibody host or isotype. However, valuable cellular material and antibodies targeting dimly expressed antigens or rare cellular populations create difficulties when using this approach. Furthermore, native cells are difficult to standardise and introduce additional variability. Compensation procedures using antibody-capturing beads overcome some of these limitations. However, many bead kits are host-specific and do not cover the full range of isotypes. In addition, high backgrounds upon violet laser or red laser excitation are observed for the vast majority of capture bead kits. As a result, Spherotech offers the COMPtrol line of antibody capture beads. The COMptrol beads offer: Low autofluorescence regardless of excitation wavelength or detection bandpass Enormous breadth of compatible hosts and isotypes makes COMPtrol capture beads a truly universal compensation tool SpheroTechnical information: Why compensation must be optimised to obtain consistent and allow for proper data interpretation for multicolour applications An introduction to Spherotech COMPtrol beads which are designed to capture antibodies with conjugated fluorophores to provide detectable signals How the COMPtrol antibody capture beads provide proper compensation values during multiple fluorophores flow cytometer experiments when combined with acquisition and analysis software STN-19: Covalent Coupling of T25 DNA to Carboxyl Magnetic Particles SPHERO T25 DNA Coated Nano Particles and Microspheres: Used for the detection and identification of oligonucleotides Allow for simple, rapid and reliable binding of Biotinylated PCR hybridised with a poly(dA)-tailed oligo SpheroTechnical information: A covalent coupling protocol for amino terminated oligonucleotides to carboxyl functionalised superparamagnetic nanoparticles using T25 DNA as an example A hybridisation procedure using T25 DNA Coated beads as an example A protocol for measuring the concentration of hybridised oligonucleotide using T25 DNA and A25-FAM DNA as an example STN-20: Activation Techniques for Hydroxyl Magnetic Particles Hydroxylic particles are used to facilitate the development of immunoassays, as well as, quantitate, isolate purify and characterise proteins, cells, or various target analytes. In order to provide the benefits of hydroxyl groups, Spherotech offers SPHERO Hydroxyethyl (HEMA)/Polystyrene Copolymeric Magnetic Particles. These copolymeric particles provide a hydrophilic surface which reduces denaturation of immobilised protein. In addition, the hydroxyethyl groups hydrogen bonds with a layer of water in an aqueous solution preventing aggregation of the particles. These two characteristics translate into longer ligand stability, lower nonspecific binding potential, and a more stable particle solution compared to particles of more hydrophobic surfaces. Although hydroxyl groups do not spontaneously react toward functional groups on biomolecules, they can be activated for covalent coupling by a number of known reaction mechanisms. The reactions result in covalent attachment of ligands and can be performed under aqueous or non-aqueous conditions. By utilising non-aqueous conditions, the hydrolysis of activating agents and the intermediate groups is prevented. The SPHEROTM HEMA/Polystyrene Copolymeric Magnetic Particles: Minimise non-specificity problems and provide stable particle solutions Can be activated using a variety of methods for the coupling of ligands in either aqueous or non-aqueous solutions Yield intermediate after activation that will then bind to a thiolated ligand or the amino groups in the ligand being conjugated Can utilise cross-linkers that serve as spacers between the bound ligand and the particle Serve to expand the repertoire of molecules capable of being conjugated to magnetic beads SpheroTechnical information: An introduction to the hydroxylic beads and their benefits A protocol for the utilisation of a maleimide-and-isocyanate crosslinker for attaching hydroxyl magnetic particles to compounds with sulfhydryl groups A protocol for the activation of hydroxyl magnetic particles using imidazole carbamestes for the spontaneous binding to proteins A protocol for the activation of hydroxyl magnetic particles using disuccinimidyl carbonate to create an NHS-carbonate particle STN-21: Streptavidin Particles Uses and Protocols Streptavidin is a protein (MW of approx. 66,000) made up of four identical subunits, each containing a high-affinity binding site for biotin (KD = 10-15 M). It has the same biotin-binding properties as avidin, but less non-specific binding is observed. It has been used both in immune assays and genomic assays for target detection. Spherotech Streptavidin bead surfaces are designed as a matrix for simple and efficient methods such as: Protein-coated beads for the isolation of biotinylated compounds such as proteins, immunoglobulins, sugars, lectins or DNA/RNA and microRNA Magnetic streptavidin-coated beads can be used as substrates for both immune assays and genomic assays Small fluorescently labelled streptavidin particles can be used as probes for detection SpheroTechnical information: Instructions for the preparations of SPHERO Streptavidin Particles before use Optimised biotinylation protocols for various ligands in order to facilitate optimal binding to An introduction to SPHERO Streptavidin Coated Beads capture antibodies with conjugated fluorophores to provide detectable signals Protocols for the immobilisation of biotinylated nucleic acids, antibodies, and PEG-biotins Methods to quantitative the amount of biotinylated DNA after binding to SPHERO Streptavidin Coated Beads STN-22: Goat anti-Mouse Particle Uses and Protocols Since the 1950s, latex-based immunoassays have been used in clinical laboratories. The first described latex agglutination assay was developed by Plotz and Singer as a Rheumatoid Factor test in 1956. Currently, in the life sciences immunoassays are used for detecting different proteins, hormones, and antibodies. Spherotech Goat anti-Mouse bead particles are designed for: The improvement of Sandwich Assay development by optimising the orientation of monoclonal antibodies for site-specific immobilisation of antigens Reducing the complexity of Magnetic Immunoprecipitation using Goat anti-Mouse IgG SpheroTechnical information: Information on how to use Spherotech antibody coated beads in sandwich immunoassays Protocols for developing a bead-based assay Protocols for washing Spherotech antibody-coated beads and coating with monoclonal antibodies Protocols for the Magnetic Immunoprecipitation using Goat anti-Mouse IgG (Fc) Magnetic Bead Protocols for regeneration and reuse of Spherotech antibody-coated beads STN-23: Magnetic Antibody Coated Particles for Cell Isolation Spheroteh Goat anti-Mouse Magnetic beads coated with a primary mouse IgG antibodies are ideal for isolation of cells from different species (e.g. human, rat) depending on the specificity of the primary antibody. Cells can be directly isolated from any sample, such as whole blood, bone marrow, MNC suspensions, or tissue digests. SpheroTechnical information: Information on how to use Spherotech antibody-coated magnetic beads for cell isolation using positive and negative selection techniques Details on the handling, washing, and storing magnetic beads used in cellular isolations Protocols for the direct and indirect isolation techniques Recommendations to improve the success of cell isolation
Spherotech Product Detail Sheets
Download Product Details Sheets: Gold Nanoparticles Rainbow Calibration Particles for Flow Cytometer Performance Verification Rainbow Calibration Particles for EuroFlow Panel Standardisation Rainbow Fluorescent Particles for Flow Cytometer Alignment & Set-Up Verification Ultra Rainbow & Supra Rainbow Quantitative Particles Kit with NIST assigned ERF (Equivalent Number of Relative Fluorospheres) Ultra Rainbow Calibration Particles for Flow Cytometer Performance Verification Ultra Rainbow Fluorescent Particles for Flow Cytometer Alignment & Set-Up Verification Supra Ultra Midrange Rainbow Fluorescent Particles for Set-Up Verification Other Fluorescent Alignment Beads for Flow Cytometry Other Calibration Beads for Flow Cytometry - Rainbow Linear, APC Channel, IR Calibration, & Blank Calibration Beads Drop Delay Calibration Particles for CCD Based FACS Cell Sorters Easy Calibration Fluorescent Particles for Flow Cytometry Quantitative Analysis Fluorescent Particle Slides Flow Cytometry Absolute Counting Particles (AccuCount) Flow Cytometry Multiplexing Assay Particles Flow Cytometry Magnetic Multiplexing Assay Particles Flow Cytometry Size Standard Kits Nano Polystyrene & Nano Fluorescent Size Standard Kits Flow Cytometry Antibody Capture Compensation Particles (COMPtrol) Flow Cytometry Surface Labelled Compensation Particles (EasyComp) Fluorescent Particles IR Fluorescent Particles Coated Fluorescent Particles Polystyrene Particles Silica Particles Functionalised Polystyrene Particles Coated Polystyrene Particles Blue and Red Coloured Particles Paramagnetic Particles Superparamagnetic Nanoparticles Silica Magnetic Particles Coated Magnetic Particles Fluorescent Magnetic Particles Coated Fluorescent Magnetic Particles Ferromagnetic Particles Coated Ferromagnetic Particles