Flow Cytometry (FCM) / FACS Antibody with Verified Stability

Antibody stability

Antibody stability or degradation can also affect staining. The immunoglobulin fraction of polyclonal antibodies is somewhat less stable than whole antiserum. This reduced stability depends largely on the method of purification, storage and application. Exposure of antibodies to extreme pH, as well as high or very low concentrations of salts, during affinity purification methods tends to decrease their stability more than does exposure to mild conditions such as ion exchange chromatography. Formation of soluble aggregates and subsequently, precipitated polymers are the most frequently resulting changes noted. These changes are probably the result of hydrophobic interaction between the IgG molecules in solution. While the presence of soluble aggregates may enhance their performance as precipitating antibodies, their increased hydrophobicity has been shown to cause increased nonspecific binding. Removal of these aggregates and polymers from IgG fractions is, therefore, important prior to applications for flow cytometry. Just as storage of purified antibodies may augment their hydrophobicity due to aggregation and polymerization, so may their conjugation to other molecules, such as fluorochromes.

Monoclonal antibodies also have been shown to be influenced in their performance by methods of purification and storage.

For example,


Antigen: Human E-CAD
Antibody: E-Cadherin / CDH1 / E-cad / CD324 Antibody (PE), Mouse MAb(10204-MM08-P)
Cell: MCF-7
Flow Cyt: Use 5 µl for 10^6 cells
Instrument: BD FACSCalibur
Antigen: Human EGFR
Antibody: EGFR / HER1 / ErbB1 Antibody (FITC), Rabbit MAb(10204-MM08-P)
Cell: A431
Flow Cyt: Use 5 µl for 10^6 cells
Instrument: BD FACSCalibur

Although our produced immunochemicals are guaranteed to be stable for up to several years, more dilute antibodies formulated for few or no washing steps have a shorter shelf life. Log in reagents by entering our lot numbers, expiration date, date of receipt and invoice number. These entries provide valuable information for the user to track their use and performance.

Storage

Perhaps the two most important considerations when storing antibodies are the storage container and the temperature. Ideally, storage containers for protein solutions should have negligible protein absorptivity. Polypropylene, polycarbonate or borosilicate glass are recommended and are used widely. Solutions containing very low concentrations of protein (i.e. less than 10–100 μg/mL) should receive an addition of bulk protein. Generally, 0.1% to 1.0% bovine albumin is used to reduce loss through polymerization and adsorption onto the container. In addition, containers made of tinted material are preferred, as these will prevent quenching of fluorochromes. Container labels also should allow access for inspection.

Immunochemical reagents should be stored in equipment with temperature alarm and emergency back-up power systems. Store most "ready to use" antibodies and their conjugates at 2–8℃ because freezing and thawing is known to have a deleterious effect on their performance. This also applies to entire kits that contain monoclonal antibodies. Store concentrated protein solutions, such as antisera and immunoglobulin fractions, in aliquots frozen at -20℃ or below to prevent cycles of repeated freezing and thawing. Bring frozen protein solutions to room temperature slowly and avoid temperatures above 25℃.

Flow Cytometry / FACS Background

Flow cytometry is a method to evaluate cell membrane proteins and intracellular proteins as well as peptides and DNA. The principle behind FACS is an antigen-antibody reaction, with the antibodies being fluorescently labelled. There are three fluorescent proteins (R-PE, APC, and PerCP) conjugated to antibodies. Flow cytometry quantification is carried out with intercalating color labels (without the antibody). Flow cytometry antibodies are widely used in cell counting, cell sorting, biomarker detection and protein engineering.