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Hello, The antibodies that I have been having difficulty with are for detecting cystathionase in HUVECs: ab54573:, XX-XX-XXXX, lot #: XXXX ab80643:, XX-XX-XXXX, lot #: XXXX The pancreatic cells had been reported to have only low amounts of cystathionase, so we instead moved on to HUVECs which are known to produce cystathionase in detectable quantities. Staining the membrane and the gel showed good transfer of proteins to the membrane. When imaging the blot with secondary antibody, I was able to see strong signal from ab54573 but only for a band present at the top of the gel .I tried digesting the protein lysate at both higher and lower temperatures and still observed the same results. I have not tried a concentration of this antibody higher than 1:500. I wasn't able to see anything on the membrane with the ab80643, trying both 1:1000 and 1:500 concentrations of antibody .Blocking was done in 5% BSA/wash buffer. Since both these antibodies should detect the same protein, I am having the most difficulty determining why the results are so different (one has strong signal albeit in the wrong position, and the other does not). Are these antibodies sensitive to the phosphorylation of the protein? I will look into trying a phosphatase inhibitor with the lysate preparation. I would like to determine whether this lysate preparation is or is not appropriate for the cystathionase protein. Would it be possible to obtain a positive control to determine whether we can detect the protein? Thank you!
Asked on Dec 14 2011
Thanks for your reply. We don’t have any evidence of the expression of cystathionase in HUVECs. If you have data to support the presence of the protein in the cell line, but not in high quantities, it would be better loading bigger amounts of sample per lane. Attending to the antibodies datasheets, for ab54573, K-562 cell lysate was used to detect Cystathionase. We do have in our catalogue K562 cell lysates that could be used as positive controls. The link to the search result is: https://www.abcam.com/index.html?pageconfig=searchresults&search=K 562&pt=12&sk=targ&sv=K562&sn=K562&l=1&fViewMore=1 For ab80643 the suggested positive control is Transfected 293T cell lysate. Unfortunately, we have 293T cell lysate in out catalogue, but not the transfected one, that would be the one recommended to work with. HeLa lysate can also be used as a positive control. There are some HeLa lysates available in the catalogue, if you wish to have a look: https://www.abcam.com/index.html?pageconfig=searchresults&search=HeLa&pt=12&sk=app&sv=69&sn=WB&l=2&fViewMore=1 I would encourage you to perform the WB again following the recommendations given (higher protein amount, using phosphatase inhibitors, concentrating the ab dilution as well as increasing the secondary concentration) and maybe using any of these lysates to check whether the problem comes from the antibody or from the protein expression level. In case you can’t notice any improvement, please contact me again and I’ll try to give a solution as soon as possible.
Answered on Dec 14 2011