Antibody Information
Antibodies
Antibody shipping
Antibodies are shipped in polystyrene igloos with refrigerant appropriate for the prevailing climate. Due to the temperature-sensitive nature of antibodies, special processing, shipping and packaging is required and, as such, additional costs may be incurred. Depending on the destination, restrictions may apply to the day we are able to dispatch. Please contact the Customer Service Department for details.
Storage and stability
Antibodies should be stored at -20°C; avoid storage in a frost-free freezer, as changes in moisture and temperature may affect stability. Stored under these conditions, the antibody will be stable for 6 months.
Blocking peptides
Blocking peptides can be used to show the specificity of antibodies as they specifically block antibody and antigen interactions in Western immunoblotting. Depending on product availability, Tocris will endeavor to provide a free sample of blocking peptide with each antibody order where listed in the catalog. Blocking peptides may also be purchased separately as catalog items, again, subject to product listing and availability.
Subject to availability, Tocris will endeavour to provide a sample of blocking peptide with each antibody supplied. Blocking peptides may also be purchased separately.
Blocking peptide use
It is recommended that the blocking peptide is used at 100-500 fold molar excess to antibody.
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Step 1
First calculate the antibody’s approximate molarity using the optimal working dilution for Western immunoblot analysis (typically 0.5 – 2 μg/ml) and an average IgG molecular weight of 150000 Da.:
| Antibody molarity |
(M) = μg (amount of antibody) |
|
150000 (average molecular weight of antibody) |
Secondly, to calculate the amount of blocking peptide (μg) required, multiply the antibody molarity by the following:
x 2 (peptide binding sites per antibody)
x 200 (fold excess peptide)
x peptide molecular weight (from product datasheet)
Example: For an antibody with optimal working concentration of 0.5 μg/ml in a final volume of 10 ml, calculate the amount of blocking peptide (molecular weight 2345) required at 200-fold excess.
Solution:
| 5 μg |
x 2 x 200 x 2345 |
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| 150000 |
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= 31.3 μg blocking peptide |
Step 2
Dilute primary antibody in 100 μl of appropriate buffer containing the amount of blocking peptide as calculated above. Incubate with gentle agitation overnight at 4°C.
Step 3
Adjust volume of antibody/blocking peptide mixture to 10 ml with appropriate buffer and analyze by Western Immunoblotting following the standard protocol.
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Antibody incubation
Many protocols incubate antibodies for 1 hour at 37°C to save time. However, we recommend an overnight incubation at 4°C to help prolong the antibody and antigen interaction and obtain an optimal signal to noise ratio. Low temperature incubations can also minimize problems with lower affinity antibodies such as those discriminating between phosphorylated and unphosphorylated forms of the target protein.
Bovine Serum Albumin (BSA)
Tocris antibodies do not posses any risk of livestock disease. The glycerol used as a freeze-thaw protectant is synthetic and most of our antibody buffers contain Trehelose rather than BSA as a stabilizing agent. Where BSA is used in a buffer, it has been sourced from countries certified as free from prion disease.
Sodium azide
Sodium azide is added to Tocris antibodies to aid long-term storage of antibodies. Sodium azide however, strongly inhibits horseradish peroxidase based detection systems. We therefore do not recommend the inclusion of sodium azide in working buffers but suggest that solutions are appropriately sterilized by either filtration through a 0.2 micron filter or autoclaved. Where horseradish peroxidase based detection systems are being used, we recommend that membranes are washed throughly prior to detection to remove any residual traces of sodium azide.
Antibody applications
Our antibodies have been tested by Western immunoblotting as a standard part of our QC procedures. However, we do not routinely check them for other applications due to the number of variables involved. We are always delighted to hear of new applications for our antibodies.
Cross-reactivity
Antibodies are assessed for species cross-reactivity between human, rat and mouse tissues and this are stated in the catalogue and datasheet entries. For alternative species we suggest the use of a protein-protein (Blastp) Blast Search [www.ncbi.nih.gov/BLAST/] to compare the protein sequence homology between the peptide sequence used in raising the antibody and the alternative species protein sequence. Although all our antibodies are affinity purified against the original peptide sequence, some cross-reactivity against proteins differing by only one or two amino acid residues within the overlapping region can be expected. We cannot guarantee that the antibody will recognize the alternative protein sequence. If the antibody does not initially recognize the alternative protein sequence we suggest removing the Tween20 detergent from all buffers and extending the 4°C incubation period to maximize potential binding.
Suggestions for antibodies
We welcome suggestions for new antibodies. Please complete your product requirements and we will contact you shortly.
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