Anti-CD45 antibody [MEM-28] (ab8216)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM no band-->even at different concentrations (range from 1:500-1:1000) SAMPLE human Hodgkin-&primary mediastinal B-cell lymphoma cell lines ( L428, L540, HDLM-2, KM-H2, L1236,MedB-1) human tonsil whole protein extract in RIPA lysis buffer PRIMARY ANTIBODY ab8216, anti CD45, mouse monoclonal tried almost every dilution between 1:500 and 1:1000 in milk or only TBST over night incubation at 4?C DETECTION METHOD pierce: super signal west dura POSITIVE AND NEGATIVE CONTROLS USED positive control: human tonsil ANTIBODY STORAGE CONDITIONS 4?C short term storage SAMPLE PREPARATION in RIPA lysis buffer NuPAGE LDS sample buffer heated for 10 Min at 95?C AMOUNT OF PROTEIN LOADED 100?g ELECTROPHORESIS/GEL CONDITIONS 4-12%Bis-Tris gel MOPS running buffer TRANSFER AND BLOCKING CONDITIONS transfer buffer (invitrogen) blocked in 5% milk SECONDARY ANTIBODY pierce - secondary anti mouse antibody (HRP conjugated) dilution: 1:1000 incubation: tried 1h at 37?C to 2,5 hours at RT HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? dilution time of incubation, temperature while incubation buffer blocking ADDITIONAL NOTES I'm a little upset this antibody does not work. CD45 is a real routine antibody for IHC and now it does not work for western blot? Please help me with my problems. I have had enough problems with abcam antibodies such as SHP-1

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with ab8216.

I have read your technical questionnaire and I have a few comments. You are using an extraction protocol that I would advise: RIPA buffer. I also consider your choice of a positive control good in the form of human tonsil.

I would not recommend using as much as 100ug in western blotting. However, given that you are obtaining no signal whatsoever, please can you tell me whether you have confirmed the successful transfer of your blot by Ponceau S. Also can you tell me whether you have confirmed the integrity of your lysate preparation using an alternative CD antiserum using these conditions.

I look forward to hearing from you.

I bought a cd45 antibody (ab8216) a few weeks ago. Unfortunately it does not work for Western Blot. The datasheet says that it should work for this technique. Could you please help me with that problem because I have already tried different concentrations and treatments, as well as the standard abcam protocol. Please answer me as soon as possible.

ANSWER:

I am sorry to hear that you have been experiencing problems with ab8216 in Western blotting. 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 90 days of purchase), and if it appears that the antibody is at fault, a credit note/refund will be offered.

Below I enclose a link to a questionnaire which will help you put your protocol information together very easily so we can look into the details of the experiment and provide some help.

Thank you, and we look foward to hearing from you.

http://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=8216&mode=questionaire

What is the epitope recognized by this antibody? Does it recognize any fragments less than the expected size of the protein? I am interested in studying these cleavage fragments.

ANSWER:

We do not know the epitope or if this antibody recognizes any fragments less than the expected size of the protein.

We do not have access to the original WB data. We did not perform such a study, and most likely, if we did and saw any smaller fragments, we would consider it as non-specific proteolytic cleavage during sample preparation rather then as a result of some biological process in a cell. Moreover, the major bands migrate between 180-220 kDa, so any cleavage fragment would presumably be lost due to the low density of gel used for LCA isoforms (so the fragments would migrate with the front of the gel).

In short, you should test this hypothesis in your particular experimental setup. If you have any additional questions, please contact us again.

Regarding a previous enquiry - could I use the mouse monoclonal antobody on paraffin-embedded mouse tissue in combination with the DAKO animal research kit (for mouse monoclonal antibodies on mouse tissue)?

Or is there any anti-CD45 antibody suitable for paraffin-embedded mouse tissue?

ANSWER:

ab8216 has not been tested in mouse, so we have no further information on this. We have several CD45 antibodies so I suggest that you conduct a search through the Abcam Website. I also recommend that you should click on the links “Product Reviews” and “Technical Enquiries” both accessible via the green menu bar on the datasheet. Here you will find customer reviews and past questions and answers specifically relating to that product.

If you cannot find what you are looking for within the Abcam catalog, I suggest that you click on the link to "The World's Antibody Gateway". The World's Antibody Gateway is a free search engine service provided by Abcam to help you to quickly find the antibodies that you are looking for. It is a free-text search engine developed by the Abcam team so that it searches the catalogs of all online antibody companies (currently 249).

I am looking for an anti-CD45 antibody that is reactive with sheep tissue (prefaerably paraffin embedded tissues).

My questions are:

1. Is ab8216 reactive with sheep tissue or is it likely to be on the basis of between species homology.

2. If not, can you recommend an antibody that does work on sheep tissue.

Many thanks

ANSWER:

This antibody has not been tested for cross reactivity with sheep. As noted on the datasheet, for immunohistochemistry, no treatment of parrafin sections neccessary. I suggest that you examine the degree of sequence homology between human and sheep CD45. We do not have an antibody agaisnt CD45 that has already been tested for cross reactivity with sheep, but I suggest that you conduct a search through the Abcam home page, and click on the link in the yellow menu bar to "The World's Antibody Gateway" to determine whether any other companies stock such an antibody. The World's Antibody Gateway is a free search engine service provided by Abcam to help you to quickly find the antibodies that you are looking for. It is a free-text search engine developed by the Abcam team so that it searches the catalogs of all online antibody companies (currently 169).