Anti-Blooms Syndrome Protein Blm antibody (ab2179)

DESCRIPTION OF THE PROBLEM Non-specific staining
SAMPLE Immortalized Human Bloom Syndrome cell line (6040 hTERT)
PRIMARY ANTIBODY ab2179 dilutions, 1:100, 1:200, and 1:400 incubated 1hr, wash: 3X 5 min in 0.2% tween-20 in pbs
DETECTION METHOD Fluorescent microscope
POSITIVE AND NEGATIVE CONTROLS USED Positive Control: BJ hTERT (WT) Negative Control: 6040 hTERT (BS line)
ANTIBODY STORAGE CONDITIONS 4C
FIXATION OF SAMPLE 4% Paraformaldehyde
PERMEABILIZATION STEP 1min in .2% Tween-20 in PBS
BLOCKING CONDITIONS Blocking Buffer: 1% BSA, 0.2% tween-20, 0.02% azide in PBS 30 min at 37C
SECONDARY ANTIBODY Invitrogen: Alexa Fluor 488 goat anti-rabbit IgG 1:400 dilution, 1hr at 37C wash: 3X 5 min in 0.2% tween-20 in pbs
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1
HAVE YOU RUN A NO PRIMARY CONTROL? Yes
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
ADDITIONAL NOTES I've attached a picture of the IF I obtained on my BS cell line (negative control) Blue: dapi, Green: Blm ab2179. I've looked through the literature and haven't been able to find anyone using any of the BLM antibodies against a BS cell line. Has this or any of the BLM antibodies been tested on Bloom Syndrome cell lines?

ANSWER:

Thank you for contacting Abcam regarding ab2179.

I am sorry that you have been experiencing difficulties with this antibody in ICC. I have reviewed the protocol information you provided and would like to make some suggestions to improve your results with this antibody.

Regarding your question about your cells, at this time we have not tested nor received feedback from anyone who has used this antibody in a Bloom Syndrome cell line. As a positive control, I would recommend testing HeLa cells treated with 15uM Bleomycin for 2 hours as this produces a positive result with the antibody.

In terms of protocol modifications, I would recommend the following:

Since this is a nuclear protein, I would recommend triton X-100 to permeabilize - try 0.1% in PBS for 10 min. If you continue to use Tween-20 I would increase this to at least 10 min. Alternatively I would recommend fixation with acetone, which will both fix and permeabilize the cells.

The blocking step seems sufficient, but I would increase the incubation with the primary antibody to at least 2 hours, although ideally overnight at 4oC.

I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or if these suggestions do not improve your results with this antibody. The antibody is guaranteed under our Abpromise for WB and I would be happy to offer a replacement or credit if we determine the antibody is not working properly.

ab2179 (Blooms Syndrome Protein Blm antibody) ab5446 (Blooms Syndrome Protein Blm antibody)

Can you provide the immunogenic peptide sequence?

ANSWER:

Thank you for your patience in this matter.

I have received a reply from the originator of ab2179 and ab5446. Although they can not provide the exact immunogenic peptide sequence (proprietary), they can provide the following information:

ab2179 - The epitope maps to a region between residue 1425 and the C-terminus (residue 1477) of human Bloom Syndrome using the numbering given in entry NP_000048.1 (GeneID 641).

ab5446 - The epitope maps to a region somewhere between residues 100 and 150 of human Bloom Syndrome using the numbering given in entry NP_000048.1 (GeneID 641).

I hope this information helps.

BATCH NUMBER 194730 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No band except background band at 50kd

SAMPLE cell extract from mouse embryonic fibroblasts

PRIMARY ANTIBODY anti-BLM 1:2000 in 3% BSA, TBS-T overnight at 4 degrees 3 washes in TBS-T 10 min. each

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED negative control (BLM RNAi) Have successfully used anti-human BLM antibodies from a different manufacturer to detect BLM in human cells

ANTIBODY STORAGE CONDITIONS 4 degrees

SAMPLE PREPARATION cell pellets are lysed in the presence of inhibitors, and sonicated cell pellets are from actively dividing cells

AMOUNT OF PROTEIN LOADED 100ug per lane (the amount needed to see a good signal from Wrn (another RecQ helicase)

ELECTROPHORESIS/GEL CONDITIONS 4-8% gradient gel

TRANSFER AND BLOCKING CONDITIONS transfer: 1 hr. 15 minutes 30v (according to Invitrogen protocol, large molecular weight bands transfer fine as seen by staining the membrane)

Blocking: 30 min. in 3% BSA, TBS-T

SECONDARY ANTIBODY anti-rabbit secondary 1 hour 1:5000 in TBST at RT 3 10 minute washes in TBST at RT

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

ANSWER:

Thank you for taking the time to complete our questionaire and send these details to us. I am very sorry to hear you have had diffficulty obtaining satisfactory results using this antibody (ab2179).

Following your enquiry, I have investigated the history of this antibody further. Mouse reactivity was added to our data sheet following positive customer feedback of results in mouse in WB. Since this time, we have had a small amount of customer feedback to indicate that the results of WB on mouse samples using this antibody are inconsistant. Some obtain just a band at 50-55kDa, some obtain both bands (55 and 170kDa). I can find no explanation for the appearance of the band at 55kDa, other than it is probably non-specific binding. Therefore, I have modified the data sheet to explain that we cannot guarantee this product in mouse samples as results are inconsistant.

I apologise for the inconvenience this must cause you. I appreciate you have put a lot of effort into these experiments. I would like to offer you a refund or credit note. If you send me your order number, and let me know how you would like to proceed, I will be pleased to arrange this for you immediately.

I look forward to hearing from you.

Dear Hugh, The blots of tubulin and Bloom antibody blots that I sent you are totally different gels with the same protein extracts. I agree that the 50-55 kd band is quite prominent, Do you have any information of any degradations that might occur to Bloom where the degradation product recognized by your antibody is a 55 kD bloom fragment.

I guess that is all I can think of, though I think we are totally out of antibody during optimizations even before we started our actual experiments. I plan to order more only once this technicality can be sorted out.

Hope to hear from you soon.

ANSWER:

Thank you for clarifying those details.

I have performed a brief search of the literature and unfortunately I have been unable to find details of Blm truncation/processing. We do not have a recommended positive control. However, given the application of the antibody to 293T and HeLa cells, these would be my recommended positive controls to detect a full length protein of 170 kDa. You western blot however shows that HeLa cells generate a product akin to your "truncated" murine product.

Given that this antibody has not behaved as detailed on our datasheet I would like to offer you a replacement vial or credit note provided this antibody was purchased within the past 90 days. I feel that a replacement vial on this occasion would not help, so would like to offer you a credit note. For me to do this please can you provide me with your purchase order details and date of purchase.

I look forward to hearing from you.

BATCH NUMBER 156329 ORDER NUMBER 60927 DESCRIPTION OF THE PROBLEM Wrong band size SAMPLE 1. Hela cell total cell extract. 2. Mouse cell line 1 extract. 3. Mouse cell line 2 extract. PRIMARY ANTIBODY Rabbit polyclonal to Bloom syndrome protein. 1: 500 (picture shown), 1:2000; 1:5000, Incubated for 2 hours at RT. Three five minute washes with TBST. DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED beta-tubulin used to check for protein expression. ANTIBODY STORAGE CONDITIONS + 4 degree C. SAMPLE PREPARATION Mammalian protein extraction buffer (Pierce) Sample diluted in Reducing SDS PAGE buffer and heated at 100OC for 5 minutes. AMOUNT OF PROTEIN LOADED 20 micro grams of total protein ELECTROPHORESIS/GEL CONDITIONS Reducing 12.5 % gel. TRANSFER AND BLOCKING CONDITIONS Transfer buffer (Methanol containing) Overnight transfer. 5% Skim milk in TBST is blocking agent. SECONDARY ANTIBODY 1:5000 of goat anti-rabbit IgG-HRP (Santa Cruz). Incubation for 1 hour at RT. Washes similarly. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Dilution of the antibody mostly. Am trying to increase the protein concenteration on gel and repeat the blot. ADDITIONAL NOTES The expected band appears slightly at 1:2000 dilution though the wrong band size (55kDa) appears more prominantly. The gel picture shown is that for 1:500 dilution of primary antibody. The control without the primary antibody did not give any background.

ANSWER:

Thank you for e-mailing me your blot images.

Once again I am sorry to hear that you are having problems with this antibody. The band that you are detecting at 55KDa is indeed very prominent.

Please excuse me if it sounds an impossible scenario but is there any chance that the antibody has been "contaminated" or mixed up with the tubulin antibody. Its just that I am aware that tubulin migrates around 50-55KDa in the same position as that located on your blot. Can you tell me whether you are probing the same membrane with the tubulin and Blm antibody or are you performing parallel experiments? If it is a strip of the membrane following a reprobe can you please tell me whether it is possible that the 55KDa band could be due to inefficient membrane stripping? The secondary antibody would detect the tubulin antibody still bound to the membrane if this is the case.

Your comments are appreciated. I look forward to your reply.