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Thanks for your reply. In answer to your questions:
2) RIPA was the lysis buffer used
3) As you can see from the blot, we tried BSA for blocking but this lead to a higher background than milk
4) I will try lower antibody dilution
5) The secondary antibody is working well with other primary antibodies with no background, including when the same blots were stripped and re-probed with GAPDH
6) Our wash steps are TBS-0.05% tween 3 x 7min after primary and secondary antibody incubation
Our main issue is there is no detectable band at 84kDa, the predicted Mw of SEMA4A, even in Jurkat cell lysate, the suggested positive control on the SEMA4A spec sheet, the strongest detectable band is at >100kDa.
I have attached another blot using, as you suggested, primary anti-SEMA4A @ 1/2000 and wash steps in 0.2% tween 4 x 5min. This lead to a much cleaner blot. However, it does not address the main question which is: human SEMA4A has a predicted Mw of 84kDa and we only see bands >100kDa.
At this stage would it be possible to get a replacement antibody? As I do not think that any more optimisation will show up bands of the predicted Mw.
Asked on Oct 23 2012
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:
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.
Answered on Oct 23 2012