Here is our western results. We try to understand these band appear like this. Do you have any opinion for solving this problem?
Okay.... seems like your stacking gel is not doing its job in creating sharp bands. Doublecheck all buffer receipts are right. Check pH (8.8 at room temperature) and molarity (0.5M tris for the 4x stock) of the stacking gel buffer. Same important is running buffer: Use Tris base ( not Tris/HCl !) and glycine. DO NOT adjust the pH of the running buffer (glycine and tris will give you the right pH on their own) as cloride ions destroy the stacking process. 1x running buffer shall contain 0.1% SDS. Last, rinse the loading pockets with running buffer and allow the gel to run slowly at the start. I start with 50-60 V until the sample is in the stacking gel, followed by 90 V until running front is in dissolving gel.
Hope that solves your problem. good luck
Okay.... seems like your stacking gel is not doing its job in creating sharp bands. Doublecheck all buffer receipts are right. Check pH (8.8 at room temperature) and molarity (0.5M tris for the 4x stock) of the stacking gel buffer. Same important is running buffer: Use Tris base ( not Tris/HCl !) and glycine. DO NOT adjust the pH of the running buffer (glycine and tris will give you the right pH on their own) as cloride ions destroy the stacking process. 1x running buffer shall contain 0.1% SDS. Last, rinse the loading pockets with running buffer and allow the gel to run slowly at the start. I start with 50-60 V until the sample is in the stacking gel, followed by 90 V until running front is in dissolving gel.
Hope that solves your problem. good luck
Agree with Christian.
also would like to add, I do not know if you are following this or not, avoid using Tris buffers prepared on the same day. Prepare the concentrations and leave overnight to stir followed by its pH check before use. I used to get similar problem unless I tried the above mentioned details. pH of buffer changes overnight and concentration cannot be assured if prepared on the same day.
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