3 replies to this topic

[yja97]

Hi. My problem is just like the title.
SDS PAGE of my protein after TCA ppt makes smear instead of discrete bands.

I precipitated my protein sample with tca + na-deoxycholate, followed by washing with acetone and resuspended with 1X SDS-PAGE loading buffer.
At first the sample is yellow, so I added a small amount of 1N NaOH until the color changed to blue.
I need  my sample for western blotting, and the signal intensity is good but the shapes are still long circle. I need to make them bands.

How do you think I can solve this problem?

Thanks in advance.

[mdfenko]

you are probably experiencing incomplete solubilization of your protein.

the yellow indicated that you still had acid in your protein. using naoh is harsh and may cause some decomposition of the protein. we use 0.5M tris (pH9 or base, whichever you prefer) to neutralize remaining acid.

both of these can cause smearing.

[yja97]

Thank you so much.
If insolubilization matters, how can I increase the solubility?  
Longer vortexing and heating would be helpful?

mdfenko, on Jun 17 2009, 11:56 AM, said:

you are probably experiencing incomplete solubilization of your protein.

the yellow indicated that you still had acid in your protein. using naoh is harsh and may cause some decomposition of the protein. we use 0.5M tris (pH9 or base, whichever you prefer) to neutralize remaining acid.

both of these can cause smearing.

[mdfenko]

yja97, on Jun 17 2009, 05:11 PM, said:

Thank you so much.
If insolubilization matters, how can I increase the solubility?  
Longer vortexing and heating would be helpful?

make sure you properly neutralize the tca then heat for an appropriate amount of time.

keep in mind that at least part of your problem was caused by using naoh to neutralize. naoh hydrolyzes proteins.