Recently, I am working on DNAPKcs phosphorylation,which is 450 kDa, I am struggling with detection in my WB(Western Blot).
First, our model organism is frog, so the first problem is the specificity of antibody.
I tried to use 5%,6% SDS gel, and add SDS in my transfer buffer. However, it seems not be working. Usually, there is no signal or a strong background that I cannot see any band.
Does anyone have some suggestions?
The key factors that can improve your results are:
1) Increase the transfer time to overnight (usually is 90 minutes) reducing the voltage
2) Reduce the methanol in the transfer buffer to 10%
3) Use NuPage antioxidant
Good luck,
Giulio
One more suggestion:
Try to position the protein in the middle of the gel. The transfer efficiency of the protein, from gel to membrane, is best when it is in the middle of the gel.
If the protein is all the way ontop of the gel, the transfer will not be efficient.
You can always stain the gel, after the transfer, to make sure you have it on the membrene for Western blot.
Good luck, Hediye.
Hello Songli,
I agree with Hediye and Giulio. Just a curiosity, do you check the proteins on your membrane post transfer with Ponceau S? And maybe a lower % gel. Also do you perform wet or dry transfer? I am more accustomed to dry transfer, and transferring large MW proteins with dry transfer, may not give the best results. In case you detect protein on Ponceau stained membrane, the problem here maybe the state of your protein...like if it aggregated while extraction and now the detection epitopes are unavailable??
Good luck!
Aparajita
Not every time, I used Ponceau S before I transfer, I haven't try dry transfer, what we have are wet transfer equipments in our lab. I think I am OK with the staining of Ponceau S.
Thank you all for your suggestions.
Hold on...Ponceau S before transfer??? Ponceau S is used on the membrane post transfer to visualize if the proteins were transferred alright, that is why I asked if you had a proper Ponceau S staining on your membrane? In case your transfer is faulty, that could explain why you do not detect your protein.
regards
Actually I mean is after not before. Sorry for that.
Sometimes I can see bands on the membrane.
You said you're not sure about the specificity of the AB so that's where I'll start. You can try doing a dot-blot or running a control lane with extract from the species the antibody was raised against.
Increasing transfer time is a good idea. I have also found that positioning the gel in the transfer apparatus so the top of the gel is at the bottom give better transfer of the large proteins.
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