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What type of beads were used for IP? *Protein G-agarose beads* > How the IP was performed? Have you mixed antibody beads and lysates once, or beads and antibody first then lysates or antibody lysates and then beads? *Antibody (6ug) with 100ul of beads o.n., 4ºc in the next day, 1mg of protein was incubated during 1h at 4ºC* >For how long the mixture was incubated? *1h* >Can we define the problem as no bands in IP or multiple bands because loading 1000 ug protein per well in WB is way too high. If it is not true then I am presuming that this is the amount of protein (lysates) you have used to mix with antibody and beads. *1mg was used to the IP for the input, 50ug of protein* > 1/1000 dilution means the antibody was used at 1ug/ml which is very low I would recommend using ab at 5 ug/ml and 10ug/ml. *1/1000 was the dilution used in the 3 abreviews that were availabe together with the datasheet. So the dilution is not the problem at least in the input lane it was supposed to see a stronger band at 37kDa and none at 50kDa.* > Could you send us an image? *Sorry, i attached a file previously, but then the page have reloaded because i've spent more than 20min filling the form. How can i send you the image? I've tried to reply by e-mail but it was not delivered... Here's a link with the image that i wanted to attach: http://imageshack.us/f/31/hnrnpa2.png/
Asked on Oct 28 2011
Thank you very much for sending the image and all the details. I acknowledge the time you have spent trying this antibody and convinced that the observed band size is not at 37 kDa. The antibody should be detecting the band at right size; the rat protein is approx 99% similar to human protein. I unfortunately can‘t compare the results with our own lab data because ab6102 was never tested with rat lysates. It may be worth going through the literature and comparing the results with other scientist’s findings. In order to proceed further in this case I can either send you a free of charge replacement of ab6102 from different lot or can also dispatch a different antibody e.g. ab31645 or ab24409 or ab64800; although these antibodies are not tested in IP however I don’t see a reason why these antibodies can’t be used in IP. If you are interested in trying these let me know I will then send you a vial of ab. I also want to point out the background in IgG IP which kind of show similar pattern as IP of A2; I am not sure why this is, the IgG IP should be clean; one reason could be the high protein conc. as compare to the antibody. I will look forward to receiving your reply soon. Please do not hesitate to contact me if you have any question.
Answered on Oct 28 2011