i run 10% native gel, as per protocol given in molecular cloning (sambrook russell book) for SDS-PAGE, except SDS and Beta ME in loading dye and staking and running gel. But could not succeed. A smear was observed. Voltage was 150. Isoelectric point of my protein is 7.3.
What do you mean by 'But could not succeed.'? Do your see a smear or the protein didn't migrate at all into the gel? What buffer is your protein in?
my protein is in phosphate buffer ph 7, 100mM Nacl. A smear was observed. Ip is isoelectric point
Guangming Chen, IP means Isoelectric point. And yes Cornelia is right. What is the problem with the gel?
We usually call it PI. In SDS-PAGE, the SDS makes the protein carry on lots of negative charge. So, the protein can run from cathode to anode.
In native PAGE, there is no SDS. We have to use buffer to make the protein carry on negative charge. You may use buffer which pH is very higher than the protein's pI.
Including protein buffer, sample buffer, gel buffer and electrophoresis buffer.
@Essak khan.. a smear of protein from top to half of the gel was observed.
@Guangming chen.. oky, that would be nice to try..thanks. i will use all the buffers of ph 8.8.
How does the gel look when you do a SDS-PAGE (denaturing conditions)? Do you see a smear as well?
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