Anti-BTK (phospho Y223) antibody (ab51210)

Is this antibody available without glycerol?

ANSWER:

Following up my message from yesterday, the laboratory tells me that they will not be able to provide this Anti-BTK (phospho Y223) antibody without glycerol. Please let me know if you have any questions or if we can help in any other way.

I would like to know what is the difference between the different lot # for this polyclonal rabbit antibody (ab51210). I have been using lot#308391 for the past month and I just reordered 3 new vials with lot#348315 which gave me a little different reactivity by Western Blot. How can I be sure that I can get consitant results with this antibody for the next year or so. I am unterested in ordering large quantities of antibody with the same lot# if possible.

ANSWER:

Thank you for your enquiry.

I have checked these lot numbers and found that they come from the same master stock reference 5119, they were just aliquoted at different times and should therefore behave in an identical manner. Could the small change in reactivity be due to the secondary, or the samples maybe? I'm afraid I do not have an explanation for this change. We still have 1 vial of lot 5119 in stock and if you require more I can try to contact the source and see if we can make a special order for you, please just let me know how much you would like. I hope this information helps. Please do not hesitate to contact us if you need any more information.

BATCH NUMBER 308391 ORDER NUMBER 243406

DESCRIPTION OF THE PROBLEM Non-specific band

SAMPLE Mouse haematopoietic cell lines Ba/F3 and 32D plus or minus Bcr-Abl expression.

PRIMARY ANTIBODY 1:1000 in TBS-T Overnight incubation.

DETECTION METHOD West Pico Pierce

POSITIVE AND NEGATIVE CONTROLS USED Bcr-Abl expression is know to phosphorylate BTK, cells plus or minus Bcr-Abl

ANTIBODY STORAGE CONDITIONS -20

SAMPLE PREPARATION 1X Cell Signalling lysis buufer. Comes as 10X 20 mM Tris-HCl (pH 7.5) 150 mM NaCl 1 mM Na2EDTA 1 mM EGTA 1% Triton 2.5 mM sodium pyrophosphate 1 mM beta-glycerophosphate 1 mM Na3VO4 1 µg/ml leupeptin

AMOUNT OF PROTEIN LOADED 100 ug

ELECTROPHORESIS/GEL CONDITIONS BIo Rad pre cast tris-HCl criterion gels,

TRANSFER AND BLOCKING CONDITIONS Transfer 300mV for 8hrs, PVDF membrane, blocked in 5% milk.

SECONDARY ANTIBODY Gibco anti-rabbit HRP, 1:2000 in TBST, 30 min, 3Xwashes for 10 min in TBST

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I have altered nothing as when I probe with the antibody against BTK itself I get a perfect band at a different size. When I use your phospho antibody I get a single band at about 58 Kd instead of 76.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been experiencing problems with ab51210 in western blot.

Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a free replacement/credit note/refund will be offered.

I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. If the suggestions do not prove to be helpful, would you please be so kind to confirm the following items in order to help me better understand the cause of the problem. If you can also provide an image that would certainly help a lot.

- Were the samples kept on ice or at 4oC at all times? Detecting a band of lower molecular weight would indicate that your protein has been digested. Please make sure that you had included sufficient protease inhibitor.

- Did you use reducing and denaturing conditions? Was SDS included in your loading buffer and migration buffer?

- Was the lysis buffer you used as strong as RIPA buffer? Please try RIPA buffer as it is the strongest and will ensure that you get all the protein of interest out.

I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me again. Also, please confirm your contact/shipping address and advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.

I look forward to hearing from you again in order to resolve the matter.