How do I resuspend my lyophilised antibody?
Reconstitute in an equivalent volume to the weight you purchased, so that the final concentration is 1 mg/ml. For example, if you purchased 100µg of antibody, resuspend in 100µl of pure distilled water. Dissolve the entire contents in water by mixing and lightly centrifuging at room temperature. It is possible to dissolve the contents in larger volume (200-500µl) but the subsequent dilution may have to be adjusted. Insoluble lipoproteins may aggregate on storage. These do not interfere with the solubility of the antibody, spin down and use the clear supernatant.
How stable are the diluted antibodies?
Antibodies diluted as directed are very stable. Months and probably years. One study (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9870851&query_hl=14) has sown extended shelf life for commercial reagents, beyond the manufacturer's expiration date. For antibodies used very diluted against low level antigens (<2K molecules/cell) is probably safer make fresh solutions monthly.
Do I need to freeze my antibody aliquots?
Each freezing cycle causes aggregation of the immunoglobulin and loss of titer. Avoid freezing aliquots that you expect to use out in the next couple of years, unless they are small aliquots which you will use completely within 1 or 2 experiments. You may freeze reference reagents in small aliquots for reference or reagents to be stored without preservative.
How should I aliquot my antibody? / Are aliquots of 10 ul okay?
The best practice is to dilute the antibody before you make aliquots to store in the freezer. We recommend customers reconstitute the vial with 100 µL of distilled or deionized water, add 900 µL of PBS or Tris buffer, and make aliquots based on intended use and freeze. One suggestion is to aliquot 10 vials with 100 µL each into small eppendorf or cryovials and store any antibody that you are not using within a week in the freezer. This allows customers to pull out aliquots to thaw as needed, thereby avoiding repeated freeze-thaw cycles. We do not recommend aliquots of less than 20 µL because tiny aliquots tend to evaporate in the freezer. If you do have a very small aliquot, use the whole thing. Don’t attempt to pipette 10 µL back out; instead rinse the tube out using a pipette and dilution buffer to be sure you get all of the antibody.Note that the recommended dilutions listed on data sheets are for vials after reconstitution (before dilution for storage).
How should I store my antibody?
The majority of our antibodies are lyophilized (freeze dried) and may be stored at room temperature for shipping and short term storage (up to one week). For longer term storage we recommend freezing as stated on the product’s data sheet.
How long should I incubate the tissue/cells with the primary antibody?
Incubation for too short will not produce adequate signal. Incubation for too long can result in negative (unspecific) staining. Since the final result of your technique can only be determined at the end of your technique when no corrections can be made, the dilution and time of incubation for each antibody should be determined individually. In general, antibodies with known high affinity should be used at high dilution and overnight incubations. Antibodies with various affinities (polyclonal) must be experimented with to determine optimum time and dilution.
Immunohistochemistry (IHC) or immunofluorescence (IF/ICC)? Which is the most sensitive method?
IHC is relatively light-insensitive and allows the visualization of tissue architecture. Autofluorescence can sometimes make IF studies impossible. IF allows for staining of the same subcellular structure with different fluorophores.
Why do I need to use a positive control and a negative control?
A positive control is needed to monitor the test validity. A negative control is needed to establish a baseline or "zero", to prove that the test is the reason for the measured effect.
How much antigen is required for antibody development?
For animal immunization, 10 mg of a peptide is required. An additional 5 mg is needed when antigen-affinity purification is performed. The peptide should be supplied in a solid format free of toxic additives such as PMSF or sodium azide. When a protein is used as antigen, 5 mg is sufficient. When the protein is in solution, the concentration should be at 1 mg/ml in a buffer solution free of detergents. When the protein is insoluble, using an aggregate is the best format instead of a solubilization buffer containing denaturing agents.
How should I send the antigen to Pepmic?
Pepmic recommends using a major carrier company to send your sample for next day delivery. Use a 15-ml conical tube or insert a smaller vial into a 50-ml conical tube to avoid loss of sample due to vial breakage. If the sample is temperature sensitive, use a styrofoam box with ice packs.
Are the projects confidential?
Yes, your projects are held in strict confidentiality. If desired, we will execute non-disclosure agreements (NDA).
How does Pepmic ensure the quality of their antibodies?
Pepmic scientists test all our products in relevant applications such as western blotting, immunoprecipitation, immunofluorescence, immunohistochemistry, flow cytometry, and chromatin immunoprecipitation. When an antibody is recommend for a particular application, it indicates that the antibody has passed rigorous application-specific testing standards. Additionally, our scientists create specialized (optimized) immunostaining protocols for individual products, saving you time and reagents. We routinely test our antibodies on multiple species including human, monkey, mouse, and rat. The high quality of our products is the most important consideration at Pepmic. If you are interested in a particular application not listed on the product webpage, please contact Pepmic technical support at info@pepmic.com for further information.
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