Hi all, I'm using a dimedone-based probe to tag proteins with specific cysteine modifications. An article I recently came across that looked at the binding efficiency of this tag under different pH, and they found that the probe was much more efficient at binding to sulfenic acid-cysteines at low pH (~5.5). Since the cysteine sulfenic acid is already very labile, I want to make sure that I have the most efficient binding conditions I can, but I'm a little concerned about using a mildly acidic lysis buffer. So, I was wondering if anyone had any advice on the use of low-pH cell lysis conditions. Would the low pH hurt my lysis, or alter the composition of the lysate (aside from possible denaturation)?
I've been trying to knockdown my protein of interest using some shRNA constructs from Origene (HuSH 29-based). I received 4 different constructs, but no single construct is able to give me more than, at most, a 30% knockdown. While I'm seeing some changes in cell viability in my knockdowns vs. scramble, I'm really hoping to get a much stronger change with a stronger knockdown. Since at least two of my constructs cause a 30% decrease, would it be okay to co-transduct viral particles containing the two constructs? I'm concerned that mixing the two viruses together is going to add confusion and complexity, but I would like to get a stronger knockdown. Would that help, or is there something else I can do to increase knockdown?
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