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Rapidly decomposing HRP substrate - (Jul/27/2015 )


I'm having some strange problems with my WBs and I was wondering if anyone had any ideas to help. I was blotting for my ß-actin loading control and noticed that the signal was rapidly reducing in seconds right in front of my eyes.


I ran SDS-PAGE and transfered using standard methods (Abcam protocol as a guide), then I blocked in 5% milk-TBST, added primary antibody at a conc of 1:5000 in 5% milk-TBST overnight 4 'C, washed in TBST, then added 1:5000 mouse HRP-conjugated secondary antibody for 1 hours at RT.  I then washed again in TBST, added HRP substrate and digitally imaged.  This is the way my group and I have been doing WBs for years and its always worked perfectly.  I've been noticing that recently our substrate of choice (Millipore Luminata Forte) has been losing the signal after a few minutes.  Where the instruction manual says it should last for an hour, I have noticed that if I take an image then leave it for 15 minutes and take another the signal will be much weaker or nonexistent.  This isn't normally a problem because my images typically take 15 seconds to 10 minutes depending on antibody.  Today I could add my substrate and put my membrane into the dock and the signal would literally last 15 seconds.  I took a 15 second image, then took another immediately after and the signal was gone.  I tried this with two other substrates, washing the membrane in between - both Life Tech Supersignal Pico, and Millipore Immobilon gave the same results.


When imaging I typically (using tweezers) take the membrane out of the staining tray and dab the corner on some paper to get excess TBST off, I then place it on a thick acetate sheet sheet and add approx 0.5-2 mL of substrate for a membrane between 30-80 cm2 membrane, this is plenty to run all over the membrane and submerge it.  I roll around the acetate sheet to get the substrate to cover the membrane for 1 minute, then pour off the excess, dab the corner with some paper, put a thin acetate sheet onto the top of it and squeeze the bubbles out, then image.  Today to get decent images I had to literally pour the substrate on the membrane around where the bands are and quickly throw it in the dock.


Does anyone have any idea whats happened?  My first thought was that the substrate is being used up too fast, but when thats happened in the past I get burning of the membrane and tell-tale 'hollow bands' where the substrate has been eaten up quickly in the centre of the band but still gives off signal on the outer parts.




:EDIT: I should add that this happened for all 5 membranes I used today.  They'd all be used at least once and then stripped, one of them has been stripped 6 times and is still going strong!


I'm not an expert in westerns, but at one point we had a similar issue with our substrate. After some time we realized an undergrad in the lab was using the same pipette to dip into the two ECL reagents so they got cross contaminated. Bought some fresh substrate and worked well after that.


Also, from Thermo's manual they say to try the following if you are getting a weak signal to see if it's your HRP:


"  **To test the activity of the system in the darkroom, prepare 1-2mL of the SuperSignal Substrate Working Solution in a clear test tube. With the lights turned off, add 1µL undiluted HRP-conjugate to the Working Solution. The solution should immediately emit a blue light that will fade over the next several minutes. If no light emission is evident, test another source of HRP to determine the root cause."


a few things to consider:


is it possible that azide somehow found its way into your substrate solution? that would kill off the hrp.


have you recently changed lots of your conjugated secondary antibody?


are your secondaries old?


is the refrigerator in which they are stored operating correctly?