I am having trouble, during development of my western blot membranes (PVDF) using the film system, the films become extermly dark when I put them in the developing solution.
Brifly I summarize my protocol,
Blocking buffer: 5% skim milk, TBST, & 1% BSA (1h at RT)
Primary antibody: Beta-actin (1:1000), dilution with TBST & 0.5% skim milk [ 4 °C overnight ]
Secondary antibody: HRP conjugated anti goat (1:10,000), dilution with TBST & 1% skim milk [ 1h at RT ]
Pierce ECL PLUS substrate (1:1) incubation 1 mins.
I herewith attatched the ugly figure.
How can I reslove this issue? Thanks in advance.
Hello Dewan.
Firstly, have you activated the PVDF membrane with methanol before use? PVDF is very hydrophobic and if you expose it with water without previous methanol treatment, PVDF does not expose its full protein binding capacity.
Secondly, when I use 5% milk as my buffer (in TBST), I don't add BSA, since BSA can cause some artefacts on the membrane.
Thirdly, did you ensure that the membrane was completely immersed in the respective buffer at all times? I never allow the membrane to be out from the buffer for more than 5 seconds as the membrane can quickly dry up, thus causing the membrane to look burnt.
I agree with Paul-Antoine Gretteau, your film most likely been exposed to light. Make sure you do the whole film development process in the dark room. Using a new batch is worth a try
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