We have expressed our protein in chloroplast when we add extraction buffer it get precipitate soon after centrifugation as this is real quick even we don't have a time to prepare sample for SDS-PAGE and Bradford. We have changed the buffers and tried Tris, HEPES, Phosphate and got some how comparable results with HEPES then we check the concentration variables of HEPES from 50mM to 100mM, EDTA from 10mM to 20mM, PEFA-BLOC (protease inhibitor) from 1 to 5mM but it does't working. We have tried TCA/Aceton precipitation, 8M urea treatment in both extraction and sample buffer but still the problem is there
Hi,
That could have something to do with oxidation of your protein; did you consider including methionine as an oxidation scaffold?
Try including polyvinylpyrrolidone in the extraction buffer to inhibit polyphenol oxidases.
Have you tried adding in 0.5mM TCEP to act a gentle reducing agent to stabilize the redox environment?
Stanley Ng i have used DTT instead of TCEP but it doesn't makes ant difference
By excluding the oxidation possibility in line with your answers...What is the composition of the extraction buffer other than the options of buffering agents? Is your target a kind of membrane protein?
İsmail Emir Akyildiz 50 mm HEPES- KOH (pH 7.5), 10 mMpotassium acetate, 5 mMmagnesium acetate, 1 mM EDTA), 1 mM (DTT), 1 Complete proteinaseinhibitor (Roche, Darmstadt, Germany) and 1% b-mercaptoethanol
protein is not a membranous it was expressed in chloroplast as a recombinant protein
Try to modify the recipe by adding non-denaturing (first) and/or denaturing detergent. Are you able to solubilize the precipitate pellet using Laemmnli SDS-PAGE sample buffer...If yes, it may indicate a high hydrophobicity dissolved by solely strong surfactants, and hence, extraction buffer should also include a bit of detergent ingredient to form micelles.
It is not unusual for heterologously expressed proteins to suffer from insolubility due to the inability to fold correctly during expression. Sometimes, it is necessary to fully denature the protein with urea or guanidine-HCl, purify it, then refold it. This can be challenging. It is often worthwhile to also explore different methods of expression to get soluble protein.
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