When I perform western blot experiments I often get inconsistent results post transfer.
What I mean is that when I look at my PVDF membrane after the transfer step I can often see the molecular marker has transfered to the PVDF membrane, I can see the colours of the dye forming a ladder. The rest of the PVDF membrane is sometimes a perfect and single colour of white. Sometimes it appears to be a uniform white that has patches/blotches of a different colour white and sometimes parts of the gel appear to be a shiny and translucent colour. Sometimes it seems as if I can even see what appears to be the protein bands marked out in this fashion or sometimes the edges of the lanes of the gel. The western blot often works no problem regardess of what I see, and normally after being soaked in the blocking buffer in an hour any of these different patches go away and become uniformly white again. Sometimes my blots do fail however and I wonder if this has any contribution.
I cant find any information on this phenomen. How unusual is this?
I always activate the PVDF membrane in methanol for at least a minute, before soaking it in thransfer buffer for ten minutes (I soak the gel in transfer buffer for the same time) before assembling the transfer sandwich and doing a semi dry transfer.
Just to be clear I am talking about the appearance of the PVDF membrane straight after the transfer step. No antibodies have been applied and the blot has not been developed.
Many years ago, when I have just learnt how to do western blot, I experienced some patterns on my membrane post transfer as well. Then I learnt that if my membrane was dried, it looked burnt. The membrane got dried up especially when I accidentally touched with my bare hands. I realized that my hands absorbed the moisture from the membrane. After that, I always use a pair of forceps to handle the membrane. I used both nitrocellulose and PVDF.
The "blotch" you see are most likely caused by a high density of protein in those regions. You don't see them after blocking since you covered most of the other region with proteins from your blocking buffer or you washed them away if you added way to much protein to your well.
If you don't always see them, it might indicate an inconsistent loading quantity. You can use dyes like Ponceau S to see your protein on your membrane, since it works well with PVDF. That way you can see if your bands are somewhat consistent between transfer. It isn't a very quantitative method when use to compare a membrane to another membrane but it would give you some idea on your transfer success.
If you have doubt about your transfer, you can dye your gel ( Coomassie blue staining for example) to see if your gel still contains proteins after your transfer.
I agree that it's a good idea to stain the filter with Ponceau S after transfer. From the patterns of the bands you can have an idea of how the transfer has worked: it should be uniformly for the entire gel; and the quality of the protein samples. You may notice patches of different colors of the filter after transfer, but as long as this does not affect your western blot results, you probably don't need to worry about. It's always advisable to carry out the gel electrophoresis and transfer carefully as @Nurul and Jean-Francois suggested.
" blotch" you mean is a result of drying during transfer procedure. If you are using a semi-dry transfer system, probably your buffer is not enought. What I am trying to say is that, during transfere, electric resistance of membrane cause heat, therefore it cause drying of membrane dot by dot.
When you block it 1 h these dots will be lost.
When you use chemi-luminecense based techniques for visualazing your result wont be affected if your protein does not stay where the dry dot is.
But if you use infrared based techniques such as li-cor, your will see these dots on your membrane.
I advice you to use "a little" more buffer during transfer.
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