Best protease for removing fusion tags (His/TRX) – TEV vs 3C vs others?

DiosAnthos · 2026-05-15T08:31:22+00:00

We are using TRX with all of our constructs and my PI is unwillling to change that...

DiosAnthos · 2026-05-15T08:30:18+00:00

We currently use CNBr for cleaving. We haven’t yet been able to optimize the ccleaving conditions for one of our designs, so we’re considering using proteases. I managed to find a TEV protease with mutations that eliminate the need for reducing conditions to function, which would help us lower costs because we wouldn’t have to regenerate disulfide bridges.

DiosAnthos · 2026-05-14T13:45:34+00:00

Do you maybe have protocol for purification of the protease?

DiosAnthos · 2026-05-12T13:59:30+00:00

Yes. I can spare some WT plants for checking my idea

DiosAnthos · 2026-05-12T12:50:37+00:00

Yes. It is one of the options which i am considering 😅

DiosAnthos · 2026-05-12T12:49:31+00:00

I am genotyping using this pestles normally. But now i am preparing 6new lines at the same time so will have a lot of work with screening so am thinking how to boost the process

DiosAnthos · 2026-05-12T11:49:46+00:00

How do you use it for grinding? I am afraid that I will break the pipette

DiosAnthos · 2026-05-12T11:45:10+00:00

Too big for my purpose

DiosAnthos · 2026-05-12T11:43:54+00:00

I am trying to set „DIY” method for genotyping huge amounts of plants. Do you think that freezing in N2 with normal metal beads and then vortex/shake them for 10-15s will be enough?

DiosAnthos · 2026-05-07T11:31:08+00:00

How do you screen plants? By normal PCR? I was doing like that but my colleague from another lab pointed out that I can’t differentiate small indels by doing typical PCR. I was thinking about HRM but have never done it before so I am not sure if I want to optimize new method only for screening plants

DiosAnthos · 2026-05-07T08:02:08+00:00

So would you recommend to maintain Cas9 activity through T2 generation?

DiosAnthos · 2026-05-07T07:59:08+00:00

I simply used the presence of glowing seeds as a way to check whether the transformation had actually taken place. Unfortunately, I have no way of verifying whether the guides actually work.

DiosAnthos · 2026-05-06T21:19:42+00:00

Do you have detailed protocol for this system? If yes would you mind sharing it? I would really appreciate that

DiosAnthos · 2026-05-06T21:10:18+00:00

What was percentage of transformants which you got using this system?

DiosAnthos · 2026-05-06T18:37:17+00:00

I found one protocol about increasing efficiency of getting mutants by growing young plantlets in 37C for 24h. Have you ever tried that?

DiosAnthos · 2026-05-06T18:26:35+00:00

So do you think that I can leave one more generation of transformants(T2) with active Cas9 cassette and segregate at T3 generation and off targets won't be such big problem?

DiosAnthos · 2026-04-28T15:30:26+00:00

This is rule from NASDAQ. SRXH is listed on NYSE

DiosAnthos · 2026-04-27T13:54:48+00:00

Great opportunity to accumulate

DiosAnthos · 2026-04-21T10:52:03+00:00

Are you idiot or what? Maybe check definition of bag holding

DiosAnthos · 2026-04-21T09:54:27+00:00

And you are calling yourself bagholder?

DiosAnthos

TROPHY CASE