Hello,
I have a question about the labelling of samples and housekeeping proteins at western blots (WB). What do you think, is it correct to get bands of samples on a WB membrane by chemiluminiscence and then use fluorescence secondary antibody for beta-actin and get fluorescence signal from this housekeeping protein and then analyze the signal as "chemiluminiscence signal of the sample at the band" / "fluorescence signal of the beta-actin at the same band"?
I know that usually the same method is used for both, the sample and the housekeeping protein. But can you combine it? Is that correct too?
I am not sure about the detection limit of the both methods. What could be possible troubles with the combination? Thank you.
The purpose of the measurement of the amount of a housekeeping protein is as an internal standard to correct for differences in the amount of protein loaded in each lane. It doesn't matter how the housekeeping protein is quantified, as long as you are sure that the signal is directly proportional to the amount of the protein. So I think it is OK to use fluorescence detection of one and chemiluminescence detection of the other.
The purpose of the measurement of the amount of a housekeeping protein is as an internal standard to correct for differences in the amount of protein loaded in each lane. It doesn't matter how the housekeeping protein is quantified, as long as you are sure that the signal is directly proportional to the amount of the protein. So I think it is OK to use fluorescence detection of one and chemiluminescence detection of the other.
I agree with dear Adam B Shapiro
In addition, the new generation of gel documenting systems enable the use of near infrared (NIR) emission range, minimizing membrane autofluorescence.
Not only have fluorophores become brighter, they have become more photostable.
Finally, an abundance of protocols and guidelines for experimental design and antibody selection may help.
Regards, Pushkin Sergey Viktorovich
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