The same dilution factor of antibody doesn't fit for the endogenous and target protein.
I am working on fish model and the primary antibody which I am using for catalase (CAT) protein needs minimum 1: 1000 dilution to get band in immunoblot. At the same time, the primary antibody for endogenous control (For me GAPDH) needs 1:10000 dilution to get band.
Now, If I use same dilution for both the protein then two different scenarios are there.
- In 1:1000 dilution - the band intensity of GAPDH is much much higher and expected to reach the saturation point.
- In 1:10000 dilution - there is no band for CAT or very very faint band.
The band intensities is different in different dilution of antibody.
So, what would be the proper antibody dilution factor to normalize the WB data?
Hi Debaprasad,
Technically no, you don't have to set a specific dilution factor for your target and control proteins as not all proteins are expressed at the same amount. I believe the aim is to achieve clean, sharp, single bands, so in my opinion, it is fine to use different dilution factors and you can play around with the dilution factors for both primary and secondary antibodies to get decent signals (e.g. keep primary Ab dilution the same but increase secondary Ab dilution for GAPDH so your control bands aren't overexposed).
If you still feel uncomfortable doing so, you can visualize your CAT blots at a longer exposure time although you might get a background with very high noise. You may also reduce the exposure time for GAPDH to just a few seconds but I doubt that will do much difference. As long as you mention your dilution factors in your write-up, I believe you can avoid confusion in communicating your findings.
i agree with the the statement of Andrew, and obviously the house keeping gene will give you the intense band as they are expressed frequently in the cell. However, the other gene express in certain condition, and the expression level is not even same.
Regards
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