The Anti-V5 Antibody allows detection of recombinant proteins containing the V5 epitope. This epitope is found in the P and V proteins of the paramyxovirus, SV5 (Southern et al., 1991). The Anti-V5 Antibody recognizes the 14 amino acid sequence:
Detailed protocols for using the antibody are in the technical manual available or in published references (Ausubel et al., 1994; Harlow & Lane, 1988).
The Anti-V5 Antibody (R960-25) is a mouse monoclonal IgG2a antibody. Anti-V5-HRP Antibody (R961-25) is prepared by crosslinking the Anti-V5 Antibody with horseradish peroxidase (HRP) using glutaraldehyde.
Sufficient antibody (50 μl) is supplied to perform 25 western immunodetections (assuming 10 ml buffer per blot).
Storage Buffer: 1X phosphate buffered saline (PBS) with 0.01% sodium azide
Storage: Store at 4ºC. For long-term storage, aliquot the antibody
and store at -20°C or -80°C. Avoid repeated freezing and
Stability: 6 months at 4ºC
Both antibodies have been tested in immunoblotting and ELISA procedures. Low background was observed using chemiluminescent or alkaline phosphatase reagents for detection. In western blot experiments with purified protein, 25 ng (for Anti-V5 Antibody) or 50 ng (for Anti-V5-HRP Antibody) of recombinant Positope™ protein (Catalog no. R900-50) gave a detectable signal using a standard immunoblotting protocol (refer to the technical manual available). The Anti-V5 Antibody has also been used successfully to immunoprecipitate fusion proteins that contain the V5 epitope.
Using chemiluminescence as the detection method, no crossreactivity is observed in bacterial lysates. In mammalian lysates, a few crossreactive proteins are observed upon overexposure of blots.
We recommend the following dilutions of the supplied antibody for these applications:
For western blots, dilute 1:5000 into PBS or Tris-Buffered Saline (TBS) containing 0.05% Tween-20 and 5% nonfat, dry milk (PBSTM or TBSTM).
For ELISA assays, serially dilute into PBSTM or TBSTM and test various dilutions to determine the best antibody dilution for your application. If you use a different buffer for washing and blocking your blots, dilute as described above with that buffer. You may use other blocking agents such as bovine serum albumin (BSA) or gelatin.
If you use azide in your HRP-conjugated antibody dilution buffers, wash the western blot or microtiter wells thoroughly before adding the color development solution. Azide inhibits HRP activity. If you use alkaline phosphatase-conjugated secondary antibody, do not use PBS. Phosphate inhibits alkaline phosphatase. Use TBS instead.
Detailed protocols for western and dot blotting, ELISA, immunoprecipitation, and immunostaining are described in the technical manual available from our website or contact Technical Support.
The Anti-V5 Antibody and the Anti-V5-HRP Antibody are functionally tested by western blot as follows:
The antibody must react specifically with 50 ng of an E. coli expressed fusion protein containing a V5 epitope. Western blots must reveal a strong signal, with no nonspecific background, after a 5-minute development with a chromogenic substrate.
The antibody must react specifically with 20 ng of an E. coli expressed fusion protein containing a V5 epitope. Western blots must reveal a strong signal, with no nonspecific background, after development with a chemiluminescent substrate followed by a 1 minute exposure to x-ray film.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New York
Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
- Southern, J. A., Young, D. F., Heaney, F., Baumgartner, W., and Randall, R. E. (1991) Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics. J. Gen. Virol. 72, 1551-1557