30 ug protein Loading -> Transfer 300mA/1h PVDF
1. 5% BSA blocking 1hr
2. 1st antibody (pBeclin-1) 0.5ug/ml dilution in 2.5% BSA, O/N
3. TBS-T wash 5/5/5/10 min
4. 2nd antibody (rabbit) 0.2 ug/ml dilution in TBS-T, 1hr
5. TBS-T wash 5/5/5/5 min
6. ECL attachment
7. develope (expose time 1~ 5 min)
In the result, I got the white bands in this protein.
How to solve problem of white band in WB?
I need to help expert.
This usually happens when there is too much protein or antibody present, since the ECL substrate is almost immediately consumed by the antibody-enzyme conjugate and there is no further light production after that point (resulting in the white bands). You can either load a lower amount of protein or dilute your antibodies further. You should also try a shorter exposure time (perhaps, 10 seconds) before developing for 1-5 minutes.
hi
i agree with malhar desai . this event occur with camera base devices .
you can run one of this options :
1- get the photo in dark room by X-ray film
2- decrease sample loading ( such as 10 ug )
3- use low sensitivity ECL
4- test with DAB staining
5- decrease your secondary antibody ( such as 0.1 ug/mL)
good luck
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