Anti-SIRT1 antibody (ab7343)

How much blocking peptide should be used with ab7343.

ANSWER:

Thank you for your enquiry. For the blocking peptide we recommend using 1 µg/ml. This should be pre-incubated with the relevant antibody for 20 minutes before adding the membrane. If you have any more questions, please contact us again.

Our customer now told me that she has probed several cell lines with another SIRT1 antibody (from Santa Cruz Biotechnology) and had no problem to detect the endogenous protein. She confirmed the specificity with SIRT1 stable transfected cell lines. She would have used your antibody AB7343 as a control to verify their first results, but unfortunately this has not worked. Now I think she feels disapointed because her workgroup has spend quite a lot of money for an antibody which is obviously quite insensitive and so is of no real use to them. Do you see a way to help them? My personal view is that we should probably send another SIRT1 antibody (from our portfolio) or give a refund. Kind regards,

ANSWER:

Thank you for your reply. We are very sorry to hear that your customer is having difficulty with this antibody.

We can offer your customer a refund or a credit note, no problem

In the past, we had to rely on our customers and external researchers to test this product since we did not have positive control. We have received very good feedback from our collaborators, thus we have been confident that this antibody works properly.

Since your customer has been working with SIRT1-stably transfected cell lines, we would like to take the opportunity to ask her for a favour. We would be very grateful if she could test the new batch of this product in her system. Please would you be so kind to forward our request to her and let us know if she would be interested.

We are looking forward to hearing from you or from her soon.

Using the ab7343 to detect endogenous human SIRT1 as well as SIRT1 overexpressed in mammalian cells (not tagged) by regular immunoblotting in whole cell extracts, we find two bands: approx. 100 kDa and approx. 80 kDa (close to the predicted size). In addition, we sometimes find a very strong high molecular weight band (approx. 160 kDa). As demonstration blot in your data sheet is not very informative, we would like you to share your experience with us (where does endogenous SIRT1 migrate on regular SDS-gels). Thanks, Martin Schuler

ANSWER:

Endogenous SIRT1 runs higher than its predicted kDa, approx. 120-130kDa. There is a lower and weaker band around 100kDa that can also be detected occasionally and that we believe to be an N-terminally truncated version of SIRT1 as there is an ATG 650 nct downstream from the start ATG. As for the 80 kDa band or the strong 160kDa we have not experienced those. It could be that these bands are specific to the cell line you are using.

please advise me data sheet of product name(SIR T1) now I want search antibody(SIR 2) I think SIR 2 and SIRT1(ab7343) were one and the same product, What Do you think about SIR 2 and your product(SIR T1)? and please send me price(SIR T1)and stock.

ANSWER:

ab7343 -- Rabbit polyclonal to Human SIRT1 is in stock and priced at GBP 225.00 for 100 µl. Two peptides were used to immunise the rabbits, one of these is available as product code ab7770 -- GADREAASSPAGEP. SIRT1 encodes a member of the sirtuin family of proteins, homologues to the yeast Sir2 protein. We do not stock antibodies raised against Sir2 protein.

Question about SIRT1 - ab7343

Just looked at the Western blot for SIRT1. I was wondering why the band seems to be twice the predicted molecular weight of the protein (approx. 80KDa).

Jessica Norman

ANSWER:

Recombinant, tagged or IVT Sirt1 proteins all migrate at this higher molecular weight. The first 100 to 150 N-terminal residues are quite rich in glutamic and aspartic acid so this maybe messes up the migration in SDS-PAGE.