Anti-Bcl2 antibody (ab7973)

Our customer would like to know the dilute titer of ab7973 for IHC (datasheet not showed). We would be thankful if you could provide this information for our customer.

ANSWER:

Thank you for contacting us.

The laboratory suggest a dilution of 1:100 when using this antibody (ab7973) in Immunohistochemistry. However, this is a guideline and will require optimisation by the end user.

I hope this information is helpful. Should the customer have any further questions, please do not hesitate to contact us again.

Thank you for your reply. I have attached one of the "better" blots I got with the 7973 antibody. I embedded it in a word.doc as sometimes the password protection on the machine prevents the images from being open on other machines. Let me if there is a problem viewing, and I can try another way. As you can see, it is hard to distingish a specific band around 26 kDa due to all the surrounding non-specific binding. To answer your question, yes, I think there is some specific binding there, I just can't tell exactly where, and it obviously is not enough to overcome all the nonspecific binding. Yes, I have considered increasing the block to 5% or greater, and have had some limited success in doing this in the past with other antibodies. But based on the lack of improvement I saw by increasing the blocking time from 1 hr to overnight (~15 hrs), I was and am convinced that the blocking step has nothing to do with these results. And it is obvious from this blot that the problem is severe nonspecific binding of the antibody, and a lack of distinguising the true band of interest for quantification. Hope this helps,

ANSWER:

Thank you for e-mailing me your blot image.

The blot image suggests to me problems in either the preparation or electrophoresis of the samples. There are no clear bands; only fuzzy smears. Can you tell me whether you have evidence that your samples were not compromised by performing a parallel immunoblot with a loading control antibody such as beta actin? This will allow you to determine whether it is your samples or the antibody that has caused the problem. I understand that you have been using another Bcl2 antibody. Did you use this on the same blot? Were your results cleaner? If so please could I see an scanned image of the blot.

I would like to recommend that you use a positive control for this antibody. We recommend HL-60 and Jurkat cell lines prepared using RIPA buffer. This will fully allow you determine whether the antibody is the cause of your problems.

Thank you for your patience in this matter. I look forward to hearing from you.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 110428

DESCRIPTION OF THE PROBLEM Multiple bands

SAMPLE Mouse prostate and liver tissue extracts

PRIMARY ANTIBODY Initially used antibody at 1:100 per recommended directions or 1:500 diluted in 0.5%/0.5% Milk/BSA in 1xPBST (0.1%Tween). Incubated with membrane overnight at 4 degrees C with gentle agitation. Then washed 4 times with gentle agitation with 1xPBST (0.1%Tween) (Quick rinse, 10 min x 5 min x 5 min).

DETECTION METHOD Bands detected using chemiluminescence (Super West Dura kit, Pierce Biotechnologies, Rockford, IL) in a Bio-Rad ChemiDoc imaging system, and band densitometry was performed using Bio-Rad Quantity One software. Dectection time was about 5 min

POSITIVE AND NEGATIVE CONTROLS USED none

ANTIBODY STORAGE CONDITIONS stored at 4 degrees C

SAMPLE PREPARATION Frozen tissue samples were powdered by using a liquid nitrogen-cooled mortar and pestle and homogenized in an ice-cold lysis buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% Deoxycholate, 0.1% SDS, 0.5% NP-40, 5 mM EDTA, 100 ?M PMSF, 0.1 ?M Okadaic acid, 1mM Orthovanadate, 50 mM HEPES pH 7.4, 3 mM MgCl2, 0.3 M Sucrose, 0.5X Protease inhibitor cocktail containing bestatin, leupeptin, and aprotinin (P2714, Sigma-Aldrich, St. Louis, MO)]. Protein content was determined using the BCA assay. Protein was diluted in 2X Laemmli buffer, boiled for 5 min at 95 degrees C, quick spin for 20 sec, and loaded onto gels.

AMOUNT OF PROTEIN LOADED 30ug for prostate; 60ug for liver. Also loaded Biorad dual-color protein ladder

ELECTROPHORESIS/GEL CONDITIONS Loaded samples onto 4?15% gradient Tris/HCl minigels (Bio-Rad Laboratories, Hercules, CA), and separated with constant current (120V) for 2 hours at 4 degrees C.

TRANSFER AND BLOCKING CONDITIONS Proteins were semi-dry transferred using a Trans-Blot SD (Bio-Rad Laboratories) to PVDF membranes for 30 min at constant current (0.6mA/per membrane) using Biorad Anode (CAPS Buffer, ddH20, Methanol) and Cathode Buffer (CAPS Buffer, ddH20, SDS). On a first run, membranes were blocked with 2%/2% Milk/BSA in 1xPBST(0.1%Tween)for 1 hour at room temperature. Due to high background, also tried blocking overnight at 4 degrees C with gentle agitation.

SECONDARY ANTIBODY Bovine anti-rabbit HRP-Conjugated Secondary Antibody (1:50,000, [a competitor])for 1 hour at RT, washed 4 times with gentle agitation with 1xPBST (0.1%Tween) (Quick rinse, 10 min x 5 min x 5 min)

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

WHAT STEPS HAVE YOU ALTERED? I have tried altering the primary antibody concentration and incubation time, as well as blocking time. Started by using 1 hour block followed by 1:100 dilution of primary overnight; this yielded poor results. I tried stripping and reprobing this membrane. First I check the membrane with only secondary to verify that the primary was gone. The I proceeded to reprobe and got very similar poor results (i.e. multple non-specific binding and background). Then tried 1:500 overnight after 1 hour block, these results were still poor. Then tried overnight block and 1:500 primary for 1 hour, and still poor results.

ADDITIONAL NOTES I have since went on to use a bcl-2 antibody from [competitor] that I borrowed from a colleague that has worked better using the same default conditions as described above.

ANSWER:

Thank you for your enquiry and for completing our technical questionnaire so comprehensively.

I am sorry to hear that you have been having difficulties with this antibody. Quality is very important to Abcam. I would greatly appreciate it if you could e-mail the blot image that you are referring to. When you refer to "multiple bands" do you think that you are detecting the antibody's endogenous target?

You are performing a 2% BSA, 2% Milk block following protein transfer. Please can you tell me whether you have considered increasing the percentage of blocking solution to 5 or even 10%. In my experience this can serve to block non-specificity and direct the antibody binding to the correct target.

I look forward to hearing from you and seeing your blot image.

Here is the customer response:

"We performed the no primary ab control and there were no bands on the gel. Just the hazy background due to a long ECL exposure.

With regard to the isolation procedure, we do not have a cellular control as we do not culture Jurkat or HeLa cells in the lab. Do you have a cell extract that we can test under our conditions?"

Note from Bryan: I'll send him some from my side.

"The extraction is done by douncing tissue several times and then sedimenting by centrifugation the membrane vs soluble fractions. The membrane fraction is enriched in mitochondria as we have analyzed several gels using proteomics (LC-MS technology). The ab7973 product recognizes a band at 28K only in the pellet fractions. As the sample was prepared for electrophoresis by boiling in SDS sample buffer containing 20 mM thiol reducing agent, it is unlikely that the many more prominent upper bands we see are due to homo or heterodimers unless they survive the above conditions. I checked the epitope sequence for Ab7973 and the mouse BCL-2 is an exact match. It appears, however, that the specificity of this product for mouse brain and spinal cord tissue samples is very poor (At high Ab dilution 1:2000, the BCL-2 band is not detectable).

Was this affinity purified against an immobilized peptide?

Did the serum show multiple band reactivity. If so, it is possible that the affinity column used for this batch (Lot 137999) may be bad.

How do we proceed."

ANSWER:

The customer is correct there should be no dimers post treatment with SDS and boiling. I would still urge the addition of protease inhibitors and the use of lysis buffer in addition to dounce homogenisation. If the customer still has problems with your control let me know and I will arrange a credit note for you.

I have a technical question. I am using the anti-Bcl2 (ab7973) in western blot. I got two major bands, one at 35 kDa and the second around 90 kDa.

Could you please let me know what is the size of Bcl2 isoform that this antibody should recognize? And what size?

ANSWER:

Thank you for your enquiry.

The predicted molecular weight for Bcl2 is 26.2KDa. However, the actual molecular weight may differ due to posttranslational modifications etc. We have received an anonymous review (click the "Abreviews" tab on the product datasheet) detailing that this antibody detects a specific band at 30kDa and faint non-specific bands at 37 and 45kDa.

I hope this information helps, please do not hesitate to contact me should you require further assistance.