I have serum samples in 50% glycerol and I want to detect by Western blots the presence of specific antigens in these serum samples? However, diluting these samples 1:2 and running on a PAGE gels doesnt seem to be good as the lanes seem distorted. What can I do about it?
If the quantity of your sample is reasonable large, you can do a dialysis or buffer exchange to get rid of the glycerol. Otherwise, if the amount of sample is limited, you can use the glycerol in your sample as that required for the loading buffer to avoid sample loss. What you need to do is to make some SDS-PAGE loading buffer without glycerol (e.g. for 1ml, 1M Tris-Cl pH 6.8 0.24 ml, 20% SDS 0.3 ml, water (glycerol replaced with water) 0.3ml, B-mercaptoethanol 0.16 ml Bromophenol blue 0.6mg). Mix your sample with 2-3 times of your self-made loading buffer should overcome the problem.
If the quantity of your sample is reasonable large, you can do a dialysis or buffer exchange to get rid of the glycerol. Otherwise, if the amount of sample is limited, you can use the glycerol in your sample as that required for the loading buffer to avoid sample loss. What you need to do is to make some SDS-PAGE loading buffer without glycerol (e.g. for 1ml, 1M Tris-Cl pH 6.8 0.24 ml, 20% SDS 0.3 ml, water (glycerol replaced with water) 0.3ml, B-mercaptoethanol 0.16 ml Bromophenol blue 0.6mg). Mix your sample with 2-3 times of your self-made loading buffer should overcome the problem.
Hello @Liu,
Thanks very much for the response. I go for the second option. Thank you once more
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