Antibody Usage
- How should I store the serum / antisera?
- For short-term storage of the working antibody or of serum that has recently arrived, 2-3 weeks at 4°C is recommended. For long-term storage, we recommend storing the serum vials at -20°C, which is sufficient for several years. Storage at -80°C is fine also, but not necessary except for very long periods of time. The purified antibody should be aliquoted into working quantities and stored according to the guidelines above. The biggest threat to antibody stability is repeated freeze/thaw cycles. To avoid this, we recommend storing ready-to-use aliquots of the antibody and thawing aliquots only as they are needed. We also recommend the use of manual defrost freezers rather than frostless freezers. For more information, please visit our Antibody Storage page.
- Why am I seeing multiple or unexpected bands on a Western with my affinity purified antibody?
- It isn’t uncommon to see multiple bands even when using affinity-purified antibody. This isn’t indicative of a problem with the antibody’s specificity. Rather, this typically occurs for one of the following reasons:
- The anti-peptide antibody recognizes a homologous protein in the sample that shares one or more epitopes with the peptide sequence.
- The native protein is a different molecular weight than previously predicted.
- The antibody is recognizing either cleaved fragments of the native protein at lower molecular weights or aggregated dimers/trimers of the native protein at higher molecular weights.
- Why am I not seeing any bands on my western when assaying with the purified antibody?
- In cases where no bands are seen (or an antibody doesn’t work in a particular assay), these are the three most common explanations:
- The peptide sequence corresponds to a non-exposed region of the native protein
- The protein’s conformation in the peptide region differs enough that the antibody has trouble recognizing the native protein.
- The target protein isn’t present in the sample.
- How do I interpret the ELISA results?
- The ELISA measures the peptide-specific immune response. Dilutions of pre-immune serum and the first production bleed are incubated in peptide-coated microwells to determine the point where the OD reading of the production bleed is 0.1 above the background (pre-immune serum). A titer of 1:50,000 to 1:100,000 indicates a good immune response.
