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Here's a brief summary of what I did so far: 1st attempt: no signal at all (1:1000) 2nd attempt, at Tom's recommendation tried 1% BSA, and the whole membrane was black (on the film) (1:500) 3rd attempt, described below, with photos attached From left to right in all the photos: Ladder-Ctrl1-Ctrl2-Treated1-Treated2-empty-empty-Treated2-Treated1-Ctrl2-Ctrl1-Ladder The left half was blocked with milk, the right half was blocked with BSA (see below) each lane contains 120ug of whole cell lysate extracted from human immortalized BEAS-2B cells (bronchial epithelial cells) ran on an "anyKD" TGX pre-cast gel from Bio-Rad per Bio-Rad recommendations used a Bio-Rad protein kaleidoscope on each end of the gel (size marked on film - pink band is 75kDa, this protein should around 85kDa) transferred to a PVDF membrane, cut the membrane in half, and blocked half with 5% milk and half with 3% BSA for one hour (both in TBST) used SATB2 antibody (ab 34735) at 1:500 overnight at 4oC (diluted Ab in milk for half, BSA for half) I snipped on a little piece of the corner of each half and did not use any primary Ab on these pieces as a control. rinsed with TBST for >2 hours total (3 rinses) used secondary goat anti-rabbit at 1:2000 (from Santa Cruz - I have used this numerous times with no problems) rinsed for 30 minutes total in TBST (3 rinses) added ECL reagent and developed for 1 minute (see film) I used a lot of protein since I used only 50ug before (when I saw nothing) - I wanted to ensure that lack of protein was not the problem. The mRNA for this protein is highly upregulated (>20x) in the treated cells and there is no literature to suggest that it is regulated at the level of mRNA, so I would have expected the band to be strongest in lanes 4,5,8, and 9. I see nothing in the milk-blocked half and 6-7 sets of bands in the BSA-blocked half. The membrane photo is inverted in color, so those strange black lines look more like tiny white bubbles on the actual membrane, but they don't seem to correlate at all to the bands on the film/gel, so I think they are unrelated to the problem. I'd appreciate your help, thanks.
Asked on Oct 26 2011
As the images are black and white, I am unable to determine which band is the 75kDa pink band you mention in your methods. Could you send me a labeled version of the blot, so that I can see where the band of interest should be. In general though, you do seem to be getting a lot of background, no matter if you use 5% milk or 3% BSA as a blocking reagent. I can only think of a few things that may be causing such high background and the presence of multiple lanes. Firstly, since you almost trebled the amount of protein loaded and then doubled the primary antibody concentration, that combination may have caused such a high noisy signal. I understand you loaded more protein, to make sure that the lack of protein was not an issue, but in case there may be too much protein was loaded. So I would suggest either reducing the amount of protein loaded and keeping the primary antibody concentration the same or vice versa. As for the multiple bands, how fresh is your lysis buffer? Also what type of protease inhibitors are you using? When you see the ladder effect in your lanes, it could be that you either need to use fresh lysis buffer or increase the concentration of protease inhibitors in your buffer. I would just like to say that under our Abpromise, we do guarantee all of our antibodies for 6 months to work as stated on the datasheet. In this case, ab34735 is guaranteed to work in western blot and on human samples. So if we are not able to able to get this antibody working successfully for you, then we will try and find another that will. I hope some of what i have said helps and please let me know if there is anything else I can do
Answered on Oct 27 2011