Anti-SIRT7 antibody (ab13682)

BATCH NUMBER 133649

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE HEK293T whole cell extract

PRIMARY ANTIBODY abcam/Rabbit/3 % non-fat milk in TBST /1:500~1:1000/2hours~overnight incubation, 30 min. washing

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Purified protein, siRNA

ANTIBODY STORAGE CONDITIONS -20C

SAMPLE PREPARATION RIPA buffer with protease inhibitors, heating sample

AMOUNT OF PROTEIN LOADED 50 ug

ELECTROPHORESIS/GEL CONDITIONS reducing gel, 10 ~ 15 %

TRANSFER AND BLOCKING CONDITIONS Tris/Glycine/MeOH/0.1%SDS transfer buffer (deeply stained by PonceauS, other antibodies were worked well), blocking agent : 3 % non-fat milk in TBST

SECONDARY ANTIBODY Jackson/Goat/3 % non-fat milk in TBST /1:1000~1:5000/1hour incubation, 30 min.~1hour washing

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? loading quantity

ADDITIONAL NOTES She purified protein in overexpression sample and loaded much quantity. And then she could see low signal.

ANSWER:

Thank you for providing more details of your customer's protocol, below are my recommendations to increase the signal:

-as discussed before the fact the customer is having the correct results with over-expressed protein reassures us that the antibody works and that in Hek293T cell lysate the protein levels are much lower. I would recommend running the positive control of human liver tissue lysate.

-check the protease inhibitors in the lysis buffer are fresh and not old and that samples are kept on ice at all times- can I also check that the samples are left to lyse for 1hr before spinning in the centrifuge (to give time for the NP40 to permeabilise and lyse the cells)?

-milk can prevent binding of the antibody to the protein and I would therefore recommend blocking the membrane in 5% BSA (30min-1hrs) and incubating the antibody in TBST only.

-please try 1:300 dilution with overnight incubation. We have used 1ug/ml (i.e 1:500) in our positive control but this needs optimisation between samples.

- use Ecl+ or even more sensitive kits (e.g. West Pico, West dura pierce kits) as ECL is not very sensitive

-check the secondary antibody is not too old and not loosing its sensitivity, if possible by trying a different secondary antibody.

I hope these suggestions will help,

Distributor forwarded complaint:

I performed western blot using your ab10140, ab13697, ab19388, ab13682, ab13860. But these antibodies not worked properly. So I want to obtain some advice.

My sample: Hek293T cell line, expressed protein from E.cole (whole, supernatant) I had obtained good results using ab13749 (Sirt1), and other antibody. So I think my western protocol is not bad.

I could see the band only when I purified SIRT in expressed E.cole, loaded an excess quantity. In my opinion, there are some problems in antibodies titer. I wonder there is any claim or good results in other researchers. Please let me know the information. And I want to know the cell line not tissue as a positive control. I await your reply.

ANSWER:

Thank you for contacting us for technical support.

The fact the customer is having the correct results with over-expressed protein reassures us that the antibody works and that in Hek293T cell lysate the protein levels are much lower. We have not had any complaints about these antibodies and they work very well in the tested positive controls.

I would recommend running the positive controls detailed on the datasheets: ab10140: human kidney cell lysate, U937 lysate ab19388: 3T3 cell lysate ab13860: no cell line has been tested- please try human testis lysate ab13697: no cell line has been tested- please try human intestine lysate

If you require further assistance can you please ask your customer to fill in our protocol questionnaires and we can look at each antibody condition; typically please check your customer incubates at the recommended dilution in TBST overnight (he/she may need to use more concentrated antibody as this depends on the tissue), please check that degradation of the protein is not occurring and other protocol details which may make a difference,