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1. Order details: * Batch number: Ab412-200 * Abcam order or Purchase order number: S060452 * Antibody storage conditions (temperature/reconstitution etc) Mouse monoclonal ?7E6?to mono and dimethyl Arginine-ChIP grade Lot:184096 Store at 4? cold box 2. Please describe the problem (high background, wrong band size, more bands, no band etc). No band 3. On what material are you testing the antibody in WB? * Species: * Cell extract or Nuclear extract: * Purified protein or Recombinant protein: We analysis the yeast(Saccharomyces cerevisiae)60S and 40S fraction, can’t see any band(Ab 2000X dilution).Then we try to use the peptide, like symmetric dimethylarginine peptide, asymmetric dimethylarginine peptide, and MAP-dimethylarginine peptide to do WB, we can’t see any positive signal either(Ab 2000X dilution).Then we try to use the control made for ourself, is the sbp1p co-express RMT1, we can see the mass data or animo acid analysis to make sure the sbp1p protein have di-methylarginine product, but it can’t see any signal in WB(Ab 500X dilution). Then we try to use the dimethylarginine control from upstate, it didn’t have any signal in our X-ray film. 3. The lysate * How much protein was loaded: * What lysis buffer was used: * What protease inhibitors were used: * What loading buffer was used: * Did you heat the samples: temperature and time: I can’t measure how mush protein in my cell lysate, or peptide, but I can see the signal in another antibody we do by ourself. In sbp1p co-expression RMT1, I loading 1ug pure sbp1p protein. 4. Electrophoresis/Gel conditions/ Transfer conditions * Reducing or non reducing gel: * Gel percentage : * Transfer conditions: We use the 4-20% tris-glycine SDS-PAGE(invitrogen).transfer condition is semi-dry,30min up to 30volt, the current is 5mA per cm2 5. Blocking conditions * Buffer: * Blocking agent: milk, BSA, serum, what percentage: * Incubation time: * Incubation temperature: In our exp, we didn’t blocking the membrane. 6. Primary Antibody * Specification (in which species was it raised against): * At what dilution(s) have you tested this antibody: * What dilution buffer was used: * Incubation time: * Incubation temperature: * What washing steps were done: I dilute 500-2000X Mouse monoclonal ?7E6?to mono and dimethyl Arginine(in your suggestion is 500X-5000X to do western blot), incubate 1hr , room temp. then wash with TBST 5min 3times 7. Secondary Antibody * Specification (in which species was it raised against)? * At what dilution(s) have you tested this antibody: * Incubation time * Wash steps: * Do you know whether the problems you are experiencing come from the secondary? 2nd antibody I use the anti-mouse HRP antibody , 5000X dilute, 30min room temp, wash 5min 3 times. We test another anti-mouse antibody, and the same 2nd antibody, we can see the signal there. So we can say the 2nd antibody didn’t have any problem. 8. Detection method ECl, ECl+, other detection method: We use the superSignal west pico detection Kit(pierce)to detect. 9. Background bands * Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): * Is the blocking step sufficient? * Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) * At what size are the bands migrating? Could they be degradation products of your target? * Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) sbp1p co-express RMT1, and the dimethylarginine control (from upstate)expose 30 min result 11. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts * How many times have you tried the Western? * Do you obtain the same results every time e.g. are background bands always in the same place? * What steps have you altered? I try about 1month, try any positive control, hope can see the signal, but I can’t. I don’t want to try another 1 month the find the positive control, so I decide to give up this antibody.
Asked on Jul 10 2006
Thank you for your enquiry. I am sorry to hear that your customer has been having difficulties with this antibody. We have received very few complaints associated with this antibody and several reviews published detailing positive feedback. I have read your technical questionaire and I have a few comments. With regards the fractions that your customer is using, can you please tell me whether they expect the fractions that they are preparing to be rich in histone proteins or proteins rich in mono and dimethyl Arginine? At this stage I would like to recommend that your customer reduces the dilution that they are applying the antibody at and increases the mass of protein. The Abreviews that we have received detail a dilution of 1/500. The dilution range that we have on our datasheet (1:500 to 1:5000) reflects the application of this antibody to nuclear or cell lysates (1:500) and histone proteins (1:5000). Histone preparations are significantly enriched for the antigen. Your customer mentioned that they performed western blotting using a di-methyl arginine peptide and a control from an alternative source. Can you tell me the peptide and the protein that you are probing as this my concern is that these proteins are perhaps not recognized by the antibody. This antibody whilst it is has been raised against Asymmetrical NG/NG-dimethyl arginine is likely to show some bias and it would be benefitical to know the proteins that they have been using as positive controls. I look forward to hearing from you.
Answered on Jul 13 2006