I found to use a wrong secondary Ab for my WB when I visualized it with ECL, can I wash with TBST for several times and incubate the right secondary Ab? Another question is why my bands are not flat? What is wrong with my electrophoresis? Thanks!
Hi Anita,
what you want is to strip your blot, for this TBST is not enough. I have two methods for this which both works:
1. solution: stripping buffer with 62,5 mM Tris HCl pH 6,7 and 100 mM ß-Mercaptoethanol and 2% SDS;
procedure: incubate the blot for 1 h at 60°C in the stripping buffer while shaking it every 20 min; wash the blot 3-4 times in 1h with a lot of TBST; then you can start the second blot with the blocking buffer;
2. procedure: wash your blot three times for 5 min with 0,2 N NaOH at room temperature; wash it three times for 5 min with H2O at room temperature; then you can start the second blot with the blocking buffer;
And for the question about your artistic bands: I would say, your samples are not homogeneous enough. You don't have a straight line, that means that all your particles in one sample don' have the same amount of energy and can't 'go' the same way at the same time. Try to be more acurate with your samples.
Dear Dr. Anita Ma
You could rinse off the ECL reagent with 2 x 10-minute washes in TBS-T (TBS + 0.3% TWEEN 20). But since you are going to use another secondary antibody, I would recommend you go the long way namely, strip and reprobe the blot.
I would suggest you consider testing a mild stripping buffer first and switch to a harsher method if this is not successful. To check whether the stripping process was successful, you could always test its effectiveness by probing with a secondary antibody alone and using chemiluminescent substrate to detect any signal. No signal detected means successful stripping.
Regarding your second question, there are a few possibilities which you could consider.
1. There is a possibility that the gel may not have been completely polymerized. There are many factors that could affect polymerization. Such as APS and TEMED (concentration and the age of the chemical), temperature (the warmer the temperature, the faster the polymerization and in air-conditioned room (below RT) it might take longer). A temperature of 23–25°C is best for polymerization for 30–45 min. To confirm that your gel has polymerized, you may store the extra acrylamide that may be left over in the beaker and wait until it is polymerized.
2. If too much heat is applied during running the polyacrylamide gel, the gel may expand, leading to such type of protein bands.
3. Finally, you may be loading too much protein sample in the wells.
Best.
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