Hi,
I am trying to screen infected plant proteomes for targets of a secreted pathogen protein. I have purified the pathogen protein using a GST-tag on an agarose resin. I am using the Pierce Plant Total Protein Extraction Kit to extract native plant proteins from control and infected plant tissues. When I wash through the extracted control (non-infected) plant proteome, my purified GST-tagged protein remains present. However, when I wash through extracted plant protein lysates from infected tissues, I lose a large amount of my purified protein. Does anyone have any idea what could be causing this? I'm assuming it may be the plant extraction buffer? But that would not explain only infected tissues lysates removing my purified protein.
Could interacting/target proteins of the GST-tagged protein be pulling them off the resin?
Thank you,
Matt
Probably glutathione concentration is high in your infected plant extracts. When you apply the protein extract to your GST-protein bound the column, you are eluting your protein. You may try using Centrifugal unit filters to remove small proteins/peptides from both extracts, that should remove glutathione from your extracts and could help improving your results. That would be suboptimal as you could remove possible targets. I would suggest you to change your purification tag for another one such as strep tag or His tag.
Hope it helps
PD: check if glutathione is related to plant response to biotic stress, because that could help you explain why this is happening to you.
Good Day,
Do you have any comfirmation that your infected cells do indeed express your GST-tagged protein?
Sebastian Lühn Yes there was confirmation that they express the tagged protein using a Rapid GST Assay Kit (Thermo).
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