Anti-SEMA4A 抗体 (ab70178)

Thank you for your email. I have more information regarding the immunogen: The antibody binds to the PSI domain. Alignment of our immunogen sequence with human SEMA4D ACC# http://www.uniprot.org/uniprot/Q92854 shows a 25% identity in an 8 residue overlap. It is unlikely that the band at the higher molecular weight is SEMA4D as the homology is low. It is more likely to be the glycosylated protein. I hope this information is helpful. Just let me know if you have further questions.

Thank you for your reply.

Kate is currently away from the office but I will try to help in your case as best I can.

Having reviewed the case I think you may be detecting the expected protein. In the very first images you shared with us it looks as if there is a band in all the 4 different lysates at around 95 kDa. There also appears to be a second band at ˜130 kDa in both the primary human melanocyte cells and SK-N-MC cell line. When you have reduced the background in your second blot, it seems the band at ˜120 kDa and ˜130 kDa has remained.

The SEMA4A protein is reported as having a sequence of ˜84 kDa, however there are also glycosylation events reported which may alter the size observed considerably. Whilst we have observed a band of ˜85 kDa in COS-7 and Jurkat cells, an additional band of ˜117 kDa was also observed. When the antibody was used in immunoprecipitation with Jurkat whole cell extract a band closer to ˜100 kDa was observed. These observed differences may be due to the glycosulation state in different samples, as well as the way in which the gel is run.

I have found reference to this in the literature:

Elizabeth P Smith et al: Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor:

http://ukpmc.ac.uk/articles/PMC3118960//reload=0;jsessionid=Wd06PA8alA25vtiO4Rfc.0

This journal states that they have observed a band of 150 kDa as well as a band of 120 kDa corresponding to the cleaved SEMA4A product.

I would therefore think it worthwhile to repeat the experiment. The background has reduced considerably, I would consider decreasing the quantity of primary human melanocyte loaded on the gel to 5 ug lane, keeping the other cell lines at 20 ug/well.

The final blot you shared, was the jurkat cell line run? If not I would try this again too as it is a good positive control to use with the antibody.

If you would like, I can see if I can provide you with the blocking peptide to see if the bands disappear when treated with it? Or if you are still not satisfied with the results I can provide you with a replacement antibody of your choice.

I hope this information has been of some help. I look forward to receiving your reply.

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.