Anti-Phosphoserine antibody (HRP) (ab9334)

Our customer sent me additional data about your email.
Check this please and he would like to receive replacement vial with different lot#.

Best regards,

ANSWER:

Thank you very much for following up with this customer. I am sorry that the results were not improve with the suggestions.

As requested, I have added a new free of charge unit of ab9334 of a different batch to your order xx

I hope the results will be good with the new vial. Please do not hesitate to contact us again should you have any questions or concerns.

I'm seeing a black blot in WB using this antibody.

ANSWER:

Thank you for your inquiry.

I am sorry to hear that you are experiencing difficulty with this antibody. Like we said on the phone, please try diluting the primary antibody more to 1:10,000 for 1 -2 hrs at RT, increasing the blocking to 1% BSA with the primary and trying regular ECL instead of the super signal.

If these protocol modifications do not work to improve the signal, please let us know and we will look into replacing or refunding the product in accordance with our Abpromise. We look forward to hearing from you.

LOT NUMBER xx ORDER NUMBER xx DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE purified protein from sigma PRIMARY ANTIBODY Concentration or dilution 1/100 dilution (2.5ug/ml) Diluent buffer 0.5% BSA in PBST Incubation time overnight (16hrs) Incubation temperature: 4 degree C wash buffer PBST Number of washes 10min RT, 3 times DETECTION METHOD LAS4000 chemiluminescence POSITIVE AND NEGATIVE CONTROLS USED Positive control Beta-casein (Sigma, C6905) Negative control not used ANTIBODY STORAGE CONDITIONS -20 degree C SAMPLE PREPARATION Lysis buffer : not used (resuspension buffer : 20mM Tris-Cl pH 8.0) Protease inhibitors: not used Phosphatase inhibitors : not used Reducing agent DTT Boiling for 5 min? yes/no yes AMOUNT OF PROTEIN LOADED 1ug/lane ELECTROPHORESIS/GEL CONDITIONS 10% Tris-glycine TRANSFER AND BLOCKING CONDITIONS Type of membrane PVDF Protein transfer verified yes (with prestained marker) Blocking agent and concentration 3% BSA, 0.1% gelatin in PBST Blocking time 2hrs Blocking temperature RT SECONDARY ANTIBODY not used HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A NO PRIMARY CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? - ADDITIONAL NOTES -

ANSWER:

Thank you for taking time to liaise with the customer and to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

I have reviewed this case and would like to confirm the following points with the customer:

1.) In the image provided, the band of the casein protein seems to have a strange migration. Has the customer tested this casein protein on another gel with the same results?

2.)As general suggestion, I can advise to keep the samples all the time on ice for detection of phosphoprotein. I do also recommend the use of phosphatase inhibitors. As I suppose however that the MS data and the gel have been performed with the same sample of casein, I do acknowledge that the casein protein has been phosphorylated and that therefore the positive control should provide a signal. See also our protocol for phosphoproteins. http://www.abcam.com/ps/pdf/protocols/Western%20blotting%20of%20phospho-proteins%20protocol.pdf

In the event thatthis antibodyhas not worked on different blots detecting the phosphorylated casein, we would be pleased to provide a free of charge replacement orcredit note.

I am looking forward to hear back from you.

I would like to use the Phosphoserine antibody HRP conjugate (ab9334)to recognize the phosphorylated serine in the following peptide: Biotin-ADVKFSFQCPG. Should this antibody work with this peptide or does it need specific surrounding amino acid? Thanks for your help.

ANSWER:

The phosphoserine antibodies were tested positive for the following phosphopeptides:

RREILSRRPpSYR RRALpSLR TPRpSP