Hello... could someone please suggest me if the plant proteins extracted using TCA Acetone method do completely dissolve in the SDS containing buffer or do they still have some residues?
If they have residues, can we still use the samples to quantify using Bradford assay and perform SDS PAGE taking the supernatant ?
Or should we dissolve them using some other buffer ? Please suggest...
It depends on how much TCA is added. Usually, 20% is used for protein precipitation and repeated washes with acetone will remove TCA residues. Please check this article below.
Article Modified TCA/acetone precipitation of plant proteins for pro...
Hi there,
Make sure that TCA is fully neutralized prior SDS/PAGE. If not sample buffer will turn yellow when mixed to the sample. If so add a few µL of 2M Tris to neutralize excess of acid and recover the blue color.
Removal of TCA by acetone washes and following SDS solubilization can present the homogenous protein solution. No problem depending on the percentages as the authors indicated. For Bradford assay generally, it is not compatible with the SDS reagent, it is ok until 4M urea experimentally or 10% CHAPS is ok also...For SDS-page after the removal of residual TCA dissolve the pellet with Leamnli buffer and perform PAGE, for protein assay I would prefer RC-DC assay kit from Bio-rad...It is based on a Lowry assay and compatible with the Leamnli buffer ingredient.
''The RC DC Protein Assay is a colorimetric assay for protein quantitation with all the functionality of the original DC Protein Assay. This assay is based on the Lowry1 assay but has been modified to be reducing agent compatible (RC) as well as detergent compatible (DC).''
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