Anti-ADAMTS4 antibody - Propeptide domain (ab45038)

LOT NUMBER -- NOT SPECIFIED -- ORDER NUMBER 8242489 DESCRIPTION OF THE PROBLEM No signal against human recombinant ADAMTS4. SAMPLE R&D systems Human recombinant ADAMTS4. PRIMARY ANTIBODY AB45038 anti-ADAMTS4 antibody at 4 ug/ml in 1% BSA in PBS for 2 hours at RT on shaker. Wash step - 3x with 175ul per well PBS with 0.05% Tween 20 using a plate washer. Dry with paper towels. DETECTION METHOD MSD T read buffer. POSITIVE AND NEGATIVE CONTROLS USED R&D systems Human recombinant ADAMTS4 with in-house ADAMTS4 antibody - positive control. R&D systems Human recombinant ADAMTS4 with no primary antibody - negative control. ANTIBODY STORAGE CONDITIONS Aliquoted at -20 degrees. TYPE OF ELISA Indirect ELISA. COATING WELL R&D systems Human recombinant ADAMTS at 1ug/ml in PBS. BLOCKING CONDITIONS 3% BSA in PBS for 1 hour at RT on shaker. SECONDARY ANTIBODY MSD goat anti-rabbit Sulfo-TAG antibody at 1 ug/ml in 1% BSA in PBS for 2 hours at RT on shaker. Wash step - 3x with 175ul per well PBS with 0.05% Tween 20 using a plate washer. Dry with paper towels. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES Does this antibody only detect the denatured form of ADAMTS4 protein? Will it recognise the native form?

ANSWER:

Thank you for contacting us.
I am sorry to hear that ab45038 is not delivering good results in ELISA.
Unfortunately, this antibody was never tested in ELISA before and it is also not guaranteed for ELISA.
ab45038 is only tested in WB under denaturing and reducing conditions. We therefore do not know if ab45038 detects native protein. Also your results suggest it does not.
I would like to suggest to run a WB as a positive control to check if the antibody behaves like stated on the datasheet.
I hope this information is helpful. Please do not hesitate to contact me again with any further questions.

Dear Tech Support,

I have the ADAMTS4 anitbody (cat #ab45038) and have a few questions. When I was reading about the antibody it says it is for the propeptide domain. Does it detect other areas of the protein? The sample blot shows bands where the activated protein would run ~64kD (this should have the propeptide domain cleaved), but the antibody seems to detect it. I was hoping to use the anitbody as a general ADAMTS4 marker (in hopes of seeing both active and inactive bands represented). Should this antibody only show the zymogen (with the propeptide domain ~90 kD) and the shed propeptide domain after activation (~29 kD)? I am seeing a band around where the active form should be (but there should not be a propeptide domain for the antibody to detect), this band is also present in the sample blot.

Any help answering these questions is truly appreciated.

ANSWER:

Thank you for contacting us. This antibody is made against a synthetic peptide derived from the propeptide domain of human ADAMTS-4. ADAMTS-4 structure contains a signal sequence that runs from residue 1-51. The propeptide domain is variously described as running from residues 52-212 or 60-181. In either case, our antibody epitope is located approximately mid-sequence in the propeptide domain. The antibody will detect intact ADAMTS-4 and the cleaved propeptide domain (with or without the signal sequence or the rest of the protein), but not the mature protein, which is thought to run from residues 213-837. ADAMTS-4 can also be truncated at the carboxyterminal end, leading to bands that contain the propeptide domain but are missing some of the carboxy portion. A generic phenomenon with the ADAMTS proteins is multiple cleavage sites for different enzymes in the amino and carboxy portions of the protein. The predicted mass for the 213-837 mature protein is about 68 kDa. Full length ADAMTS-4 is predicted to be 90.2 kDa, and the ADAMTS-4 with intact propeptide domain and without the signal sequence is predicted to be 84.6 kDa. This does not take into consideration the glycosylation and other post-translational modifications to ADAMTS-4. Most people see ADAMTS-4 in a variety of molecular weight forms from the high 60’s top low 30’s. So, if you are seeing ADAMTS-4 at 68 kDa in a reduced SDS PAGE gel Western blot, using this antibody, it must be cleaved at the carboxy end. I am pasting below below a citation for the carboxy-cleavage of ADAMTS-4. Please let me know if you have any further questions.

Preston Alexander Arthritis Rheum. 2007 Sep;56(9):3010-9. Low molecular weight isoforms of the aggrecanases are responsible for the cytokine-induced proteolysis of aggrecan in a porcine chondrocyte culture system. Powell AJ, Little CB, Hughes CE. Source Cardiff University, Cardiff, UK. Abstract OBJECTIVE: The major proteases responsible for aggrecan turnover in articular cartilage are the aggrecanases (ADAMTS-4 and ADAMTS-5). Although several studies have demonstrated C-terminal truncation of these aggrecanases, the mechanism and importance of this processing are poorly understood. The objective of this study was to further investigate ADAMTS-4 and ADAMTS-5 C-terminal truncation in a porcine model in vitro culture system. METHODS: Chondrocyte-agarose cultures with well-established extracellular matrices were treated with or without interleukin-1 (IL-1), for a variety of different culture time periods. Cultures were analyzed for release of sulfated glycosaminoglycan, aggrecanase-generated interglobular domain (IGD)-aggrecan cleavage, and the presence of ADAMTS-4 and ADAMTS-5 isoforms. Inhibition of aggrecanase activity with monoclonal antibodies, tissue inhibitor of metalloproteinases 3 (TIMP-3), and cycloheximide pretreatment were used to identify ADAMTS isoforms involved in IGD-aggrecan catabolism. RESULTS: Multiple isoforms, including possible zymogens, of ADAMTS-4 and ADAMTS-5 were sequestered within the extracellular matrix formed by 3-week chondrocyte-agarose cultures. IL-1 exposure induced production of a low molecular weight (37 kd) isoform of ADAMTS-4. This isoform was capable of degrading exogenous aggrecan at the IGD-aggrecanase site, was inhibited by TIMP-3, was blocked after preincubation with an antibody to a sequence in the catalytic domain of ADAMTS-4, and required de novo synthesis in the presence of IL-1 for its generation. CONCLUSION: In porcine chondrocyte-agarose cultures, a 37-kd ADAMTS-4 isoform appears to be the major matrix protease responsible for the IGD-aggrecanase activity detected in response to exposure to IL-1. This conclusion contradicts that of recent studies of transgenic knockout mice and highlights the need to determine the roles of the different aggrecanase(s) in human disease