My protein are of 30-60 kD range. I use 9% gels and transfer them on Amersham's Semi dry unit (0.8mA/cm.sq for 1hr). I use 14.4g Glycine, 3.03 g Tris base and 0.1% SDS per litre of MQ water. My transfer buffer has the same composition of tris and glycine; with 0.03% SDS and 20% methanol.
All of the sudden protein stopped transferring to NC. None of the solutions were changed. The top lid of our semi dry unit had salt sediments, so I cleaned it with 10% acetic acid. After that, the protein transferred but not as good as it used to be. All of my other lab mates run 1.5 mm gels. Hence they prefer total wet transfer in a Bio-rad unit(80V/1h). I run 1 mm gels. Hence, I assumed that my transfer will also work well with the same parameters. But, majority of my protein did not transfer as was the case with Semi-dry unit.
Clearly, there is no issue with these transfer units - I tried changing all the buffer components but my problem is not rectified. I am sure I am not handling something well, but I am not sure what it is.
One quick test, before I start to speculate. Have you stained one the gels, where the transfer didn't work, after the blotting? Simply put it in Coomassie and see if you get bands. If you still get bands after transfer you definitely know that your tranfer didn't work. If the gel is blank, than something else went wrong.
SDS interferes with protein binding to NC membranes. Omit SDS from transfer buffer..!! Good Luck..!!
@ Gaber and Christian... I did stain the gels with coomassie... That is how I realised my tansfer was not working
@Goodwin .... complete wet transfer buffer did not have any SDS... It was 14.4g Glycine and 3.03g Tris-Base in 1L MQ water.
I am guessing there is an issue with my sample loading buffer.. So, I am planning to treat the cells again and make fresh samples.. I hope that works.. Thanks for your suggestions :)
@Deepa: Is anyone else using the transfer system? We once had an issue with a broken elektrode which sometimes was sometimes connected and sometimes not, depending how the lid was closed. The power supply used did give a warning when there was no current flowing, so probably check this as well.
If you want maximum sensitivity or want the results to be either internally consistent or reproducible from run to run, you need to perform wet transfers. It will give you a more-or-less quantitative transfer. Semidry is a quick-and-dirty method for very abundant proteins and non-demanding applications where you just want a quick size estimate.
I have run 1mm gels on semi-dry transfer. My transfer buffer had much less glycine than yours (5.82g Tris, 2.93g Glycine, and 0.375g SGS with 200ml methanol, brought up to one liter). Also, with semidry you may be running it too fast. On my system, running over 18V for 50 minutes you may be losing your protein. I agree with the other commentors, stain your gel with coomassie, and double check your membrane with Ponceau S.
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