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We recently purchased an Abcam antibody ab70178 toward SEMA4A.
SEMA4A has a predicted Mw of 84kDa. We have consistently seen a band at 100kDa and no band at ˜84kDa using a range of antibody dilutions, blocking conditions and human cells lines, see attached powerpoint containing representative blots.
Could you please advise on this matter.
Asked on Oct 16 2012
Thank you for taking the time to contact us with these details. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.
The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.
I would like to reassure you that ab70178 is tested and covered by our 6 month guarantee for use in WB and human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.
Reviewing this case, I would like to offer some suggestions to help optimise the results. I would also appreciate if you can confirm some further details:
1. Please provide the order number and date of purchase
2. Please confirm which lysis buffer was used? I can recommend to try RIPA if this has not already been used. This should provide a suitable protein preparation. The sample should be reduced and denatured before running on a reducing gel.
3. I can suggest to try BSA rather than milk to block, and not to mix blocking agents through the experiment. This can sometimes help to improve results, particularly if the blot is very dark.
4. Try the antibody at a lower dilution to reduce the background, try 1::2000.
5. Is the current vial of secondary antibody working well with other primary antibodies? Try a no primary control. The concentration of secondary may need to be reduced in order to help optimize the results.
6. I can recommend to include some wash steps if this has not already been tried. Wash in PBS containing 0.2% Tween 4 times for 5 minutes at each appropriate wash step. This should help to wash away any excess antibody.
I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.
Answered on Oct 16 2012