I am performing TCA precipitation to concentrate neuronal lysate fractions for SDS-PAGE then Western Blot. After I precipitate my protein overnight at 4C in 20%v/v TCA and washed with 90% Acetone twice I rehydrated the sample (0.5ug/uL) in 1X Lam buffer (with BME) and heated at 95C for 5 minutes. When I applied the samples to the gel they remained in the well but the loading dye (Bromophenol blue) still migrated. The samples are not too acidic because the Bromophenol blue is not changing colors. Would anyone know how to optimize this? Thank you!
try to neutralized your protein sample after rehydrated using NaOH and after that start your SDS-PAGE
The amount of protein in the pellet may have been too large to be solubilized by the sample buffer. Try increasing the amount of sample buffer, or using a more concentrated sample buffer to dissolve the pellets.
Alternatively, consider whether you can prepare the samples for SDS-PAGE without TCA precipitation. Use a 4X sample buffer with the original samples to minimize the dilution.
After heating the sample at 95C, Centrifuge the sample and check whether it forms a pellet? If you observe pellet that means your sample is not getting solubilized properly in sample buffer. You may also increase time of heating and/percentage of SDS in sample buffer( 4X. as suggested by Adam B Shapiro) if you observe pellete or viscus sample.
One can combine TCA with desoxycholate as carrier (doi:10.1016/0003-2697(77)90043-4), but in my hands chloroform/methanol precipitation (doi:10.1016/0003-2697(84)90782-6) gives much better results. I'd also reduce the temperature in sample buffer to 60 °C for 15 min, gentle does it.
Did you stain the gel to see if any protein had entered?
I checked via centrifugeation after solubization as you suggest Jasim and Gopal. No pellet. My loading dye is not changing colors and contains bromophenol blue (ph indication). Wouldn't that suggest there is no need for adding NaOH?
4x sample buffer didn't fix the issue because my wells are still a bit too small.
Plan: Evaluate method as described by Englebert; isolate more sample with less lysis buffer to increase overall concentration then use 4x LD if needed.
Thank all for your help I really appreciate it.
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