Anti-Phosphoserine antibody [3C171] (ab17465)

Can you provide me with an image of this antibody being used in Western blot? It will help me decide to purchase the antibody. I am not convinced without an image.

ANSWER:

Thank you for your enquiry.

I was able to obtain an image. I'll send it to you in a separate email as I cannot attach it here. You will see that the antibody was tested at 1:200 in a Drosophila melanogaster whole tissue lysate (40ug/lane). There are multiple bands as there are multiple serine phosphorylated proteins. I will add the image to the online datasheet as soon as I have more confirmation on the possible identity of these bands.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

I am looking for for information regarding cross-reactivity of this antibody, as well as information regarding its affinity for the ligand.

ANSWER:

Thank you for your enquiry.

I have updated the specificity statement for ab17465 with the following information from the source of the antibody:

Recognizes phosphorylated serine both as free amino acid and when coupled to carriers such as BSA or KLH. Does not crossreact with non-phosphorylated serine, phosphothreonine, phosphotyrosine, AMP or ATP. Species crossreactivity: Human, monkey, bovine, canine, rat, chicken, rat kangaroo, goose and hamster.

Apparently the antigen specificity is quite high; but it is an ascites grade antibody with antibody concentration indeterminant. The affinity must not be extraordinarily high with the suggested dilution of 1:250 for Western Blotting.

This is the most exact information I could find. It appears that the affinity of the antibody for its ligand was not calculated.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

BATCH NUMBER 104636 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No signal, despite having negative and positive controls on western.

SAMPLE Immunoprecipitated proteins from H1299 cell extracts transiently-transfected with proteins of interest. I can confirm that immunoprecipitation worked.

PRIMARY ANTIBODY abcam - ab17465, general phospho-serine antibody/ mouse/ PBS/3%Marvel / overnight / wash in water and PBS/0.05%tween

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Recombinant protein +/- phosphorylation by recombinant kinase.

ANTIBODY STORAGE CONDITIONS Aliquoted and stored at -20

SAMPLE PREPARATION Cell lysis with protease inhibitors and phosphatase inhibitors/ sample buffer/ heat samples

ELECTROPHORESIS/GEL CONDITIONS Reducing conditions. 7.5% gels

TRANSFER AND BLOCKING CONDITIONS Transfer onto nitrocellulose. 150mA for 1 hour. Block in PBS/3%Marvel for 1 hour

SECONDARY ANTIBODY [a competitor]/rabbit anti-mouse/PBS-Marvel 3%/1:2000/1 hour

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Thought about pre-treating cells before lysis with a Ser phosphatase inhibitor such as Okadaic acid.

ANSWER:

I'm sorry to hear you are having problems with ab17465.

Could you please clarify what you mean with "no signal despite positive controls on western"? Did the antibody work on cell lysate in WB and stop working when your protein of interest was immunoprecipitated?

Could it be that your protein of interest is not phosphorylated sufficiently to detect it by WB or total levels of the protein are very low?

It is also possible that the phosphate groups are destroyed by phosphatases. Could you tell me more about the concentrations of your phosphatase inhibitors? I would recommend making sure the samples are on ice at all times and that following stimulation the cells are snap frozen to keep the protein in the phosphorylated state. If you would like to inhibit endogenous phosphatases the addition of okadaic acid should be done prior to stimulation rather than prior to lysis, but in my experience cells can suffer a lot from this chemical.

The primary antibody should be used at a 1:250 dilution, as you have not detailed which dilution you have tested could you please confirm you have used this dilution? I would also recommend using TBST (tween20 0.1%) to dilute antibodies and to wash, as the water wash step may affect the antibody binding properties.

Finally, I would recommend ECL+ rather than ECL as this is more sensitive,

I hope these first suggestions will help you and look forward to hearing from you to help you more,

Customer Question: Please send me the sequence of KLH peptide against which this antibody has been raised. I am interested in Phosphoserine antibody having serine followed by proline and arginine.

ANSWER:

The immunogen was phosphoserine-KLH and the antibody recognizes free phosphoserine or phosphoserine conjugated to a carrier such as KLH or BSA.

There is no immunizing peptide; just a phosphorylated amino acid.