What Western blot conditions to apply for high abundance proteins?

In case of a too high protein load/well, protein bands can be observed with the naked eye, as seen in the upper image to the right. When this happens, it is a clear sign that the protein load per well has to be considerably decreased, and other parameters have to be adjusted.

Applied protocol:
Samples 1-5 are Arabidopsis thaliana transgenic lines 1-4, control 5)
20 µg total protein/well extracted with 10% TCA/Acetone.
Primary antibody: 1: 5000 1h/RT (AS03 037, Agrisera)
Secondary antibody: 1: 25 000 1h/RT (AS09 602, Agrisera)
Development: AgriseraECLSuperBright (extreme low femtogram detection range)

Protocol optimization:
1-2 µg of total protein/lane
Primary antibody dilution: 1: 10 000 1h/RT (AS03 037, Agrisera)
Secondary antibody: 1: 50 000 1h/RT (AS09 602, Agrisera)
Development: AgriseraECLBright (mid picogram detection range)

Western blot results were provided by Dr. Yordan Muhovski, CRA-W, Blegium

To save sample, primary and secondary antibodies, the protocol can be optimized and provide clean bands of a target protein. Anti-RbcL antibodies (AS03 037) have high titer, which combined with a high abundance of the target protein requires low protein load/well and high primary antibody dilution.

What Western blot conditions to apply for high abundance proteins?

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