Question (19637) | Anti-CEBP Alpha/CEBPA antibody (ab15048)

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Question

Our customer had some question about ab15048 antibody. 1. The immungen which you mentioned is NM_012524 that could be wrong. Becouse of the seqence is 1389 bp, it could't be recognized by ab15048 on 466-831 amino acid. http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=81295399 2.They encountered some trouble in WB that the data showed many non-specific bands. Could you please check these conditions below? 1. Order details: a.. Batch number: b.. Abcam order or Purchase order number: ab15048 c.. Antibody storage conditions (temperature/reconstitution etc) -20? 2. Please describe the problem (high background, wrong band size, more bands, no band etc). Many non-specific bands and I don’t know which band is target. 3. On what material are you testing the antibody in WB? · Species: mouse NIH/3T3 cell and 3T3-L1 cell · Cell extract or Nuclear extract: cell extract · Purified protein or Recombinant protein: neither, total cell extract as sample 3. The lysate a.. How much protein was loaded: 35 ug per lane b.. What lysis buffer was used: RIPA c.. What protease inhibitors were used: cocktail (pancreas extract, pronase, thermolysin, chymotrypsin, trypsin, papain) d.. What loading buffer was used: 5X sample buffer (625mM Tris-HCl pH6.8, 25% glycerol, 2% SDS, Bromopheol blue, 5% 2-ME) e.. Did you heat the samples: temperature and time: 97 ? 10min 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: Reducing b.. Gel percentage : 12% c.. Transfer conditions: 100V 4? 1hr 5. Blocking conditions a.. Buffer: PBST (0.1% Tween 20 in PBS) b.. Blocking agent: milk, BSA, serum, what percentage: 5% milk c.. Incubation time: 4? O/N d.. Incubation temperature: RT 6. Primary Antibody a.. Specification (in which species was it raised against): rabbit polyclonal Ab b.. At what dilution(s) have you tested this antibody: 1/1000 c.. What dilution buffer was used: 5% milk in PBST d.. Incubation time: O/N e.. Incubation temperature: 4? f.. What washing steps were done: 3 times each for 10 min wash with PBST 7. Secondary Antibody a.. Specification (in which species was it raised against)? Goat anti- rabbit IgG conjugated HRP b.. At what dilution(s) have you tested this antibody: 1/5000 c.. Incubation time: 1hr d.. Wash steps: 3 times each for 10 min with PBST e.. Do you know whether the problems you are experiencing come from the secondary? No 8. Detection method ECl, ECl+, other detection method: ECl 9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no · Is the blocking step sufficient? I have tested 4? O/N Yes · Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) I have washed 4 times and no difference · At what size are the bands migrating? No, because there are many bands and it’s not migrating bands. · Could they be degradation products of your target? There are non-specific bands at large size. · Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 1 n 2p 3n 4p 11. Did you apply positive and negative controls along with the samples? Please specify. Yes, lane 1 and 3 are negative and lane 2 and 4 are positive controls 10. Optimization attempts · How many times have you tried the Western? 5 times · Do you obtain the same results every time e.g. are background bands always in the same place? YES · What steps have you altered? First, Prolong blocking time, 4? O/N Second, dilute the primary Ab with PBST or 5% skim milk in PBST. Third, take the liver and fat as positive controls but there are no target bands at all.

Answer

I have received the following feedback from the originator of ab15048: According to the original researcher, the "Rabbit anti-C/EBP alpha antibody was created against c/EBP alpha recombinant protein, derived from nucleotide sequence 466-813 of rat origin (NM_012524)" This antibody should detect bands at ~30 and ~43 kDa, which your customer is detecting among the other, non-specific bands. According to the original researcher, "HeLa cells (80% confluent) were treated with trichostatin A (5 micromolar for 4 hrs) or 10 micromolar sodium butyrate for 3 hrs before harvesting and preparing for lysates by treating with SDS sample buffer (direct lysates). The lysates can be sonicated and heated (boiling) for 5 mins. SDS-PAGE and Western blot may then proceed." This preparation will help disrupt multimers, which may be causing the additional bands of higher MW so please recommend this preparation to the customer. Performing a secondary only control (no primary) will help determine if the non-specific banding is caused by an interaction with the secondary. Also, it may be beneficial to decrease the amount of secondary used. Please suggest to decrease the primary incubation time from O/N to ~1 hr at room temp. Washing of unbound antibodies may be insufficient. Increase wash periods and add 0.5% TWEEN 20 to wash solution. Also increase the concentration of salt. Increase the number of washes. Washes should be performed for at least 30 minutes changing every 5 minutes, then increase salt (500 mMol) and TWEEN 20 (0.5 to 1.0%) Here are other protocol tips to help: -It may help to try loading less protein. We generally recommend loading 10-30ug of protein depending on the samples. -Increase the detergent (i.e. Tween-20) concentration to 0.5% in your blocking buffer. -Finally, bands larger than the target protein may be due to phosphorylation, glycosylation, splicing variants and additional cross-reactivity with known isoforms. I hope that this information is helpful to your customer. Please let me know if they continue to experience difficulties.