Anti-RACGAP1/MGCRACGAP抗体(ab2270)

Thank you for your e-mail. The lysate used was Human Testis lysate lysed in RIPA buffer, the recipe for that buffer is as detailed below: 50mM Tris HCl pH 8 150 mM NaCl 1% NP-40 0.5% sodium Deoxycholate 0.1% SDS protease inhibitor cocktail (Roche or Sigma) I do not think the lower band can be the phosphorylated protein as phosphorylated proteins have relatively the same MW as non phosphorylated (if not they are a little higher but this is often not detectable by western blotting) and I think the lower band is more likely to be a degradation product (if your protease inhibitors are not efficient enough (not enough or too old)) or a fragment from post translational cleavage. I would recommend to run the positive control to see if you observe that band, and also to run a no primary control and to try blocking with BSA 5% 1 hour in case it is the blocking agent creating this problem. If you can also try overnight blocking in milk this may also be a good modification to try (if you have plenty of samples). Please let me know how you get on with those modifications and do not hesitate to contact me if you need further assistance,

Thank you for your enquiry. This antibody has been tested on Human Testis lysate, therefore we would strongly suggest using this type of lysate as positive control along with the samples. Should your customer still have difficulty with this product, then please provide some more details of his assay.