Anti-SAP155抗体(ab39578)

Thank you for contacting us.  The primary UniProt entry for this product is O75533 (this entry is reviewed).  The other two UniProt entries refer to fragments of the same protein.  We expect SAP155 to run at 146 kDa, but is possible that an additional band may be observed just above 146 kDa corresponding to the heavily phosphorylated form of the protein.  It appears that all of the SAP155 present in the HeLa cells is fully phosphorylated.  We are unsure of the identity of the weaker lower band, but it appears to be nonspecific.  I hope this helps, please let me know if you need any additional information.   

Thank you for your reply and for sending the customer's enquiry.

It may be possible to treat the lysate with a more concentrated CIP solution, however we have found that the recommended concentration tends to give the best results. I searched through some literature to see if I could find a reference of someone using concentrated CIP, but I could not find any definitive answer. I would recommend treating the samples with CIP at the recommended 1 ug protein in 10 uL of buffer, and then centrifuging the samples and aspirating some of the buffer in order to concentrate the samples before adding Laemmli buffer and loading on the gel.

I hope this will be useful, but please let me know if the customer has any further questions and I'll be happy to help. Have a great day!

Thank you for contacting us with your customer's questions. I'm hoping that I can provide some helpful responses.

1) Regarding the lysate treatment, I think it would be fine to freeze the samples prior to CIP treatment. Just lyse the cells as usual (but with a lysis buffer that does not contain sodium orthovanadate or EDTA) and freeze the lysate. Thaw when ready and proceed with the de-phosphorylation protocol.

2) Itseems that the secondpart of theprotocolis missing a few steps, so I apologize for this error.I recommend adding1-2% CIPto the CIP bufferand incubating the membrane overnightat 37 degrees C. After this incubation,wash the membrane threetimes, then proceed with theWestern blot protocol (starting with the proteinblock step).

I hope this information will be useful, but please let me know if there are any further questions and I'll be happy to help.

Thanks for getting back to me, and I'm very glad to hear that the customer was satisfied with the previous suggestions.

Every phosphatase will phosphorylate specific amino acids in proteins, and PP1 and PP2A are specific serine/threonine phosphatases. The SAP155 proteinis known to be phosphorylated at several serine and threonine residues (http://www.uniprot.org/uniprot/O75533), so PP1 and PP2A should be good phosphatases to use with this protein.

Unfortunately we don't carry phosphatases in our catalog at this time, so I will suggest searching on http://www.biocompare.com for a vendor who can provide these. Treatment can probably be applied directly to the cells in culture or to the cell lysate, but I am not sure of this and will also recommend checking with the phosphatase vendor for a complete protocol.

PMSF is an inhibitor of serine and cysteine proteases, and sodium orthovanadate is an inhibitor only of tyrosine phosphatases, so these are probably not the most relevant for the customer's study. I would suggest sodium fluoride, which is an inhibitor of serine/threonine phosphatases, or a phosphatase inhibitor cocktail which is available from other vendors as well.

I hope this information will be helpful to the customer, but please let me know if you have any additional questions and I'll be happy to help!

Thank you for your reply. My colleague Jackie is out of the office but I am happy to help you with this case. Unfortunately we have not done any further characterization of the bands that appear in the Jurkat nuclear lysate on our website. There are several known phosphorylation sites for SAP155, and the doublet in the image that you sent likely represents different phosphorylation states- http://www.uniprot.org/uniprot/O75533 I agree with Jackie that the lowest band is most likely non-specific, as there are some very faint non-specific bands on the blot on our website (though at different molecular weights than the lowest band on your blot). What kind of phosphorylation characterization studies has the customer performed? If the bands are due to phosphorylation, then sufficient treatment with phosphatases should remove the extra bands. If the customer is unhappy with the results, we will of course replace or credit/refund the original purchase. Please let me know if you have any further questions or if there is anything else that we can do for you.