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I have a query about an antibody we bought from Abcam and would be grateful if you could direct me to someone (if not yourself) who can help with this query. The antibody concerned is: Anti-ADAMTS7 antibody - Spacer region, Cat No ab28557. On the Abcam website, it is said that the antibody was generated using a synthetic peptide based on the carboxyterminal end of human ADAMTS7 between amino acids 890-950. If this is case, the antibody will be expected to detect human ADAMTS7 isoform 1 (on UniProt) which is of about 184 kDa (if unglycosylated) and consists of 1,686 amino acids. However, in our Western blotting with this antibody and whole cell abstracts, the main and largest band was about 95 kDa. We wondered whether this band is from human ADAMTS7 isoform 2 (on UniProt) which is about 95 kDa with 864 amino acids. However, this isoform does not have amino acids 890-950 and therefore would not be expected to be detected by an antibody generated from a peptide corresponding to amino acids 890-950. So, we are puzzled and wonder whether or not the information “synthetic peptide based on the carboxyterminal end of human ADAMTS7 between amino acids 890-950” on the Abcam website is correct. Could you please help us with this query? Also, can Abcam send us a Western blot image for this antibody, if available? Many thanks for your help. Best wishes
Asked on Sep 12 2011
Thank you for your feedback. I am sorry to hear you have experienced problems with ab28557. The quality of our products is extremely important to us, so thank you for bringing this to our attention. I can confirm that the immunogen is a "synthetic peptide based on the carboxyterminal end of human ADAMTS7 between amino acids 890-950." Therefore it should only detect isoform 1. I am not sure why a band at a lower size (95 kDa)should be detected. Could this be isolated to the type of samples tested? For example, in certian cancer cell lines, sometimes proteins can become truncated due to a high level of passaging and differential expression. Unfortunately we do no have an image of this antibody in western blot. I do have a list of suitable positive controls: HFL-1 (human fetal lung fibroblast) cell lysates RD (rhabdomyosarcoma, embryonal, epithelial) cell lysates Mouse pancreas tissue lysate Would it be possible to find out some more ifnormation about the protocol? I have attached some questions below. I look forward to your reply. Problem Choose: Non-specific band Multiple bands No signal or weak signal High background Lot number Purchase order number or preferably Abcam order number: General Information Antibody storage conditions (temperature/reconstitution etc) Description of the problem (high background, wrong band size, more bands, no band etc.) Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) Amount of protein loaded Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Detection method (ECL, ECLPlus etc.) Positive and negative controls used (please specify) Optimization attempts (problem solving) How many times have you tried the Western? Have you run a "No Primary" control? Yes No Do you obtain the same results every time? Yes No e.g. are the background bands always in the same place? What steps have you altered? Additional Notes: Image: We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.
Answered on Sep 12 2011