western on VEGF... I see no bands at all... ned help - (Apr/19/2007 )
hi... i'm doing Western blotting for VEGF at the moment, using the VEGF (sc-7269) antibody isoform 165 from Santa Cruz. I used MDA-MB-231 cell lysate to detect VEGF. However, not a single band was seen. Anyone here mind sharing with me what's the amount of protein u loaded for elecrophoresis? How did u go about till u get the nice single band? I used 30ug and the dilution i used were 1:200, 1:400, 1:500, 1:800, 1:1000. I've yet to see any bands yet... My I know what is the size of VEGF protein u detected?
do you use a control f.i. pure VEGF, or are you sure that the Ab works?
No. I don;t have a control.. i just assume that i'll work since many have been using it...
I have been using the same antibody for more than two years, and the problem is from Santa Cruz biotech: the first antibody I purchased worked well for immunohistochemistry and bad for western blot; the second one (from a different lot number) worked really well for both the techniques, and the third one does´t work again. In any case, the antibody recognizes mainly the high MW forms of VEGF (VEGF189 and high MW precursors), and VEGF165 and 121 can be detected only if you load a high amount of protein in your WB. For LNCaP cells, I had to load more than 50 micrograms of total protein, and more than 20 ng of purified VEGF165. For brain tumours (and with the second lot number) I had to use 70 micrograms. Actually, I am tryng with the policlonal antibody from Santa Cruz. I think that the solution is to try another antibody.
I'm now trying with the replaced VEGF mAb from SAnta Cruz, still I couldn;t get any band at all!!! In fact, I was also given polyclonal Ab for VEGF from Santa Cruz but also the same thing, no bands at all. I used 25ug of proteins. I don't know if this is too little or what? i can't afford to use higher amount coz the amount i loaded now is rough'y 30ul in each well. Should I load any more, i'm afraid it may overflow. Another thing is if I were to choose a new Ab foe VEGF, do u have any recommendations? Any Ab suits recognize both human and mouse VEGF Ab? I'm interested in VEGF 165. Have u heard of VEGF 164 by R&D?