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My protocol is the following: Blocking with TBS-Tween 5% milk for 1 hour 2*5min washes with TBS-T 1:1000 anti-NFE2L2 in TBS-Tween 5% milk overnight at 4°C 3*15min washes with TBS-T 1:10000 anti-rabbit HRP for 1 hour at room temperature 3*15min washes with TBS-T ECL
Asked on May 22 2013
After review of your protocol, at this time, I would recommend use of 3-5% BSA in TBST blocking solution or to use a casein block. There are two other possibilities which could help as well. The first is that there may be too much protein per well. I do not see that you have included this in your protocol but overloading of the gel is one of the most common reasons for non specific bands. Immobilized proteins may provide a concentrated adsorbtive surface to which certain IgG may bind nonspecifically. Similarly, such nonspecific binding may be uncovered when highly sensitive detection systems such as enhanced chemoluminescence are employed. A dilution series of the starting material usually clarifies the signals.. A second alternative is that the concentration of antigen too low. The resolution of SDS-PAGE is limited. If the relative concentration of the antigen of interest is too low (less than 0.2% of total protein), it may be difficult to detect. Signal enhancement may then lead to the appearance of artificial bands.As such I would recommend to increase antibody concentration to 1/500.
Answered on May 22 2013