hi i have been honing my skills in western blot over the past month and have been getting faint bands despite loading 100ug total protein, and varying levels of antibody dilutions. my ponceu staining turns out 'fine' for my 100ug but i have been noticing that the protein ladder (can be used as a gauge of my samples in other lanes) have been disappearing gradually by the time i detect my housekeeping protein in actin.
first day: run SDS page, western blot, blocking in milk for 1 hr, probe for protein of interest using primary antibody overnight at 4 degrees.
second day: wash in tbst 3 times 5 mins each. incubate in secondary antibody that binds to primary antibody, wash in tbst 3 times 5 mins each, block in milk 5% for 1 hr, probe for actin using primary antibody for 2 hrs RT, wash in tbst 3 times 5 mins each, incubate in secondary antibody that reacts against primary antibody 1 hr----> then detect using ECL substrate.
what i notice is as i progress throughout these steps, my protein ladder bands get fainter and fainter. why is this and does this mean my protein of interest lanes are also 'degrading'?
I have seen the ladder wash away when using too much detergent in TBST, (I routinely use 0.05% Tween or 0.1% Tween max) or when someone accidentally uses Triton instead of Tween in the TBST.
First of all you don't need to block in between the 2 antibody steps, this is redundant unless you are stripping. What is the target you are looking at, because a 100µg of protein is a lot to load for a western blot.
The marker bands getting "fainter" is a common thing. The protein marker is prestained and I believe you just wash away some of this staining and not the actual protein
I would like to add that proteins that are transferred to nitrocellulose membrane do not "degrade". However, certain proteins get "released" from the membrane with washing and storing of the membrane in TBST with time.
Hi Iwan Zaki, What Divaker Choubey was saying is that the proteins that you transferred on to the membrane may get 'released' from the membrane with prolonged storage, depending on the size and the amount amount of proteins.
the norm is 50 ng of proteins for western blot is plenty.
I would recommend checking your buffer system, as I have encounter the wash off effect of my membrane resulted from the incorrect pH, I notice my 'All Blue' protein ladder get fainter by the end of washing steps.
throw out you old buffer, and make fresh and correct pH.
Inherent to nitrocellulose (and not PVDF), blots should remain in wet condition to "retain" proteins. Generally, nitrocellulose has a lower binding capacity (80-100 ug/cm2) than other blotting membranes such as PVDF (100 -300 ug/cm2), The nitrocellulose's blot surface does not bind/retain protein that are weakly associated (reduced signal observed). Also, PVDF blots can "dry" for long term storage.
You may try to vary the conditions based on the answers you have received so far. I advice you to try first on control samples with actin and then proceed with your experiment once you have adjusted the conditions. You might also try to use dot blot first at the first optimization step then proceed with Western blot analysis.
I have seen the ladder wash away when using too much detergent in TBST, (I routinely use 0.05% Tween or 0.1% Tween max) or when someone accidentally uses Triton instead of Tween in the TBST.
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