I am performing western blot and sometimes the marker band and protein wash away after washing with TBST. I am using the same TBST and all the procedures. But I have tried with different PVDF membrane and nitrocellulose membrane (From some leftovers in the lab)
Hello Jyoti Rustagi
There could be two possibilities:
1. Protein lost from the membrane during detection.
After transfer is complete, air-dry your membrane completely for 10 mins at 37 degree C before you block. Drying the membrane allows proteins to bind tightly to the membrane, preventing potential signal loss.
If you are using PVDF membrane, be sure to activate the membrane in methanol for 1-2 mins, then equilibrate in either water or your transfer buffer.
To ensure complete transfer with maximum binding and low background, the nitrocellulose membrane, however, must be thoroughly wet in DI water (no methanol to be used) prior to transfer.
2. Protein not retained on the membrane during transfer.
SDS in transfer buffer may interfere with binding of transferred proteins, especially for low molecular weight proteins. Try reducing or eliminating SDS. However, presence of up to 0.05% SDS does improve transfer efficiency of some proteins.
Transfer of large proteins (>140 kDa) often requires lower methanol concentrations (10%) and possibly the addition of SDS to 0.05%. To get complete transfer of larger proteins, extend transfer times.
Small proteins may pass through the membrane during transfer, this process is called “blow-through”. To get good membrane retention of these smaller proteins, use higher methanol concentrations (30 – 40%) and lower voltage transfer.
You also made a mention about some leftovers in the lab.
Both PVDF and nitrocellulose membranes should be stored properly to ensure maximum shelf life. They should be stored at RT ( approximately 15-25 degree C) away from chemical vapors, which may partially dissolve the membranes thereby disrupting the pore structure, and also out of direct sunlight as it may dry out the membranes.
The shelf life for PVDF membrane in normal storage conditions is around 5 years and for nitrocellulose, not very sure, but it should be around 3-4 years. Just check the expiry dates for the PVDF and NC membranes. Also, if you have noticed that the two sides of the membrane are different. One side is rough and the other side is smooth. The proteins should be transferred on to the smooth/shiny side of the membrane.
Best.
Malcolm Nobre after blotting I was able to see the complete transfer of the stained marker bands, but later on, washing with TBST they get lost. But yesterday when i increased the incubation of x-ray film on membrane like 10 min i was able to see the band develop.
Hi Jyoti Rustagi
It is possible that the washing conditions are too harsh or the membranes are over-washed, causing the marker band and protein to wash away. It is also possible that the membrane is not properly activated before transferring the protein, leading to poor adhesion of the protein to the membrane. Here are some tips to avoid this issue:
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