Question (24758) | Anti-GEMC1 antibody (ab112571)

Go to datasheet (ab112571)

Question

I have completed the questions below and I have attached an image of our blot. I look forward to receiving any further information you may have on these antibodies. Yours Order Details Antibody code: 111178 and 112571 Problem Choose: No signal  and / or incorrect size band 111178 Lot number 111178  GR52439-1 Lot number 112571  GR52498-1   Purchase order number 60098125 or preferably Abcam order number: General Information Antibody storage conditions (temperature/reconstitution etc) kept +4 Description of the problem (high background, wrong band size, more bands, no band etc.) Incorrect size band Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) Recombinant protein and 3T3 cell line Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) Cell line boiled in 2% SDS gel loading buffer Amount of protein loaded Cell extract - 12 µg Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) Reducing and 15% Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) 2 Hour transfer 25mM Tris 200mM Glycine 0.01% SDS Blocked in Non Fat Milk PBS Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) anti GEMC1 Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Anti Rabbit HRP diluted 1/2000 (DAKO) Detection method (ECL, ECLPlus etc.) ECL Positive and negative controls used (please specify) Rabbit anti cdk 2 antibody Optimization attempts (problem solving) How many times have you tried the Western? Once Have you run a "No Primary" control? No  but controls have been used Do you obtain the same results every time? Yes No e.g. are the background bands always in the same place? N/A What steps have you altered? Additional Notes:

Answer

Thank you for taking the time to complete our questionnaire and send the images, I appreciate your time. The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality. Reviewing the results from ab112571, the blot looks as we would expect it to with a band at about 40 kDa. I am sorry I have not yet received a response from our source regarding the testing on heart lysate. I will let you know as soon as I have further information. The results from ab11178 are concerning as this has only a band at 70 kDa. The response from our source regarding testing in kidney lysate is that as far as they are aware, GEMC1 is known to be expressed at a reasonable level in kidney. I am sorry they currently have no further images to provide. I would like to offer some suggestions to help optimise the results from this antibody. I would also appreciate if you can confirm some further details: 1. I can recommend to aliquot and store the antibody at -20oC as directed on the datasheet. This will ensure the stability of the antibody is maintained and that we can continue to provide our Abpromise guarantee. 2. It is possible the 70 kD band may be a dimer and I can recommend to ensure the sample is adequately reduced and denatured. The SDS buffer should contain beta mercaptoethanol and be boiled for at least 5 minutes if this has not already been tried. 3. I can suggest to use a suitable lysis buffer if you have not already done so. I can recommend to lyse the cells in RIPA buffer before adding SDS samples buffer and boiling. SDS loading buffer alone will not necessarily lyse the cells and/or provide a suitable protein preparation. 4. We recommend to load 20 - 30 ug of total protein per lane of the gel from whole cell lysate. For purified protein, this should be 10 - 100 ng. 5. Regarding the recombinant protein samples, there are inherent difficulties with antibody detection of recombinant proteins that I can recommend considering. This may be a biological artifact rather than a problem with the antibody. Could you confirm that the protein transfected is full length and includes the immunogen sequence? Also, if the recombinant GEMC1 is tagged, it is possible that the tag is preventing access to the antibody. Could you confirm at which region of the protein the tag has been placed? 6. Could you confirm the species from which the recombinant GEMC1 gene was taken? 7. What dilution of antibody was used and how long for? I can recommend to try the antibody at 1:250 overnight at 4oC if not already tried. 8. I can recommend to try a loading control in order to assess the quality of the sample. As previously discussed, 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 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund. I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

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