I stained a nuclear protein (Lamin b1) to control the purity of my samples and the extraction of the nuclear compartment after a nucleo/cytoplasm extraction and it looks nice, but I stained also white bands in the cytoplasmatic samples (there is not a contamination because the band are several and different). The concentration of the primary is 1:500 (1:1000 it is not enough) because the first time I used it the antibody works well (and I didn't see any white bands) while the secondary concentration is 1:5000. This primary antibody has been used for the second time
White bands are usually where the label is totally consumed. Cut back on either the primary or secondary Ab.
I have experienced white bands too when I increased the concentration of the antibodies.
Try to get back to dilution suggested by the manufacturer. Probe primary ab overnight at 4C and washing step after that is actually crucial. Try 5 times 5 min washing with 1 x TBST. and prob secondary ab for 1 hr at RT. again washing carefully.
As for the ECL, I used only 1ml per membrane, where I pippete the ECL solution over the membrane (gently) for 5 min before viewing under the GelDoc.
Play around and I am sure you will get your own trick on western blotting :) Gambatte!
Hello Marco,
Ghost bands often appear when the concentration of antibodies is too much and the HRP is consuming fast and concentrate the waste of ECL reagent in your bands. Try to decrease the antibodies and try to set up again the time exposition of the films.
Its not bad if you check again the pH of your buffers, could give problems with developed.
Hope I help you :)
Try doing the secondary at room temp for 30-45 minutes followed by washing 4 times atleast. Also use TBST with 0.5% tween. Helps!!
Check your ECL solution. Make a fresh solution and use for developing. But before that wash with 1xTBST (with 0.1% tween-20) and re-develop the blot by using fresh ECL dilution.
- Badges
- Science method
- Similar topics
- Methodology
- Laboratory
- Laboratory Techniques
- Blotting Techniques