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1) Abcam product code ab39155 2) Abcam order reference number or product batch number 3) Description of the problem detected bands differfrom the expected molcula weight of the molecule 4) Sample preparation: Type of sample (whole cell lysates, fraction, recombinant protein…):whole ell lysate Lysis buffer RIPA buffer Protease inhibitors: yes Phosphatase inhibitors yes Reducing agent yes Boiling for ≥5 min? yes 5 min Protein loaded ug/lane or cells/lane 50ug/lane Positive control yes heart cell lysate Negative control no 5) Percentage of gel 12% Type of membrane nc Protein transfer verified yes Blocking agent and concentration TBS/tween0.1%+milk0.05% Blocking time 1min Blocking temperature room temperature 6) Primary antibody (If more than one was used, describe in “additional notes”) :ab39155 Concentration or dilution 1:5000 and 1: 10000 Diluent buffer TBS/tween0.1%+milk0.05% Incubation time 10min (snap millipore) Incubation temperature: room temperature 7) Secondary antibody: anti-rabbit IgG (sc2054) Species:goat Reacts against: rabbit Concentration or dilution 1:2000 Diluent buffer TBS/tween0.1%+milk0.05% Incubation time 10min (snap millipore) Incubation temperature: room temperature Fluorochrome or enzyme conjugate:hrp 8) Washing after primary and secondary antibodies:yes Buffer TBS/tween0.1% Number of washes 3 9)Detection method chemiluminescence 10) How many times have you run this staining?1 Do you obtain the same results every time? What steps have you altered to try and optimize the use of this antibody?
Asked on Dec 20 2011
Thank you for taking the time to provide us with the further details. I apologize for the delay of my reply. I am very sorry, that you are experiencing problems with two of our antibodies. I do actually suspect that you have no signal of Adam12 and the bands at 50kD are unspecific. Indeed, I am not aware of any isoform of adam12 around that molecular weight. On Swissprot, there are several isoformes described, all however above 50kD. http://www.uniprot.org/uniprot/O43184 I am therefore sorry not being able to help you to îdentify this band. I can recommend however to add protease inhibitors to the samples in order to exclude that these bands might be degradation products. I have looked throught the details provided, which will also enable us to investigate this case and will provide us with vital information for monitoring product quality. Having reviewed this case, I would like to offer some suggestions to help optimize the results from these two antibodies. I would also appreciate if you can confirm some further details. In the event that we can not solve the problems with these suggestions (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund. 1.) I have noticed that the blocking has been performed in a 0.05% Milk solution. I suppose this has been done for one hour. I can strongly recommend to test also another blocking solution, such as 5% BSA. Also, I do normally recommend to use also a 5% milk solution. Indeed, changing the blocking solution can drastically influence and improve the results. Please see the image on the datasheet of ab9385 as an example. Click here (or use the following: https://www.abcam.com/index.html?datasheet=9385). 2.) I can also suggest to use the primary antibodies in concentration of 1/1000. 3.) As mentinned above, I can recommend to include protease inhibitors in the samples if this has not yet been done sone. If the ab29431 has been used, can you please confirm that the lysat has been stored correctly? Thank you! 4,) I would also apprecaite if you can confirm the quality of the secondary antibody: Does the secondary antibody alone not give such a similar background? Does the secondary antibody work well with other primary antibodies? I hope this information is helpful, and I thank you for your cooperation. I am looking forward to hear back from you with your comments and/or results.
Answered on Dec 20 2011