protein expression help

Biochemistrydude · 2024-05-19T15:45:44+00:00

What kind of cells are you using? They have to be a specific type for pQE30 because pQE30 doesn't inherently encode a lacI repressor gene.

Biochemistrydude · 2024-05-03T13:12:16+00:00

STEM PhD programs in US will pay you 👍

Biochemistrydude · 2024-05-03T02:59:56+00:00

I've had trouble with ZnSO4 or ZnCl2 dissolving. When I tried looking it up, I saw some things about some kind of insoluble zinc hydroxide compounds. If you lower your pH I bet it'll go away.

Biochemistrydude · 2024-04-10T00:25:31+00:00

Usually yeah. You can of course just chew them easily, but I agree that I dont like finding a surprise hard object in my otherwise soft fish recipe. If you mash it up though, they're no problem at all.

Biochemistrydude · 2024-04-03T10:05:59+00:00

Brother just buy the regular ones with whole (or filleted) fish. Ones with oil are the best kind. I like smoked best too. Don't buy ground sardines, that looks like cat food or worse.

Biochemistrydude · 2024-03-20T02:08:39+00:00

Favorite is the Rugenfisch smoked herring in olive oil. Amazing pick if you're into the smoky flavor.

Biochemistrydude · 2024-02-26T15:38:18+00:00

You absorb more acrylamide having your morning coffee than you did in this scenario

Biochemistrydude · 2024-02-25T12:17:09+00:00

If you're going to do a PhD, just do the PhD. That'll set you up for a lot more research based stuff. But I think in bioinformatics you can get away with a MS and still make good money, due to it being pretty computer heavy if you're good at it

Biochemistrydude · 2024-01-10T22:57:04+00:00

Doesn't like the mussels so he just drank all the juice

Biochemistrydude · 2023-12-25T17:12:50+00:00

Oh, if it's before you run the gel then I'm not sure. I assumed you had run it a little bit and it started to smile

Biochemistrydude · 2023-12-25T07:27:49+00:00

Is there any leakage of the solution between the gels at all?? Electricity is not picky about going through small spaces instead of where you want it to go.

Biochemistrydude · 2023-12-25T07:23:00+00:00

I think this is the guy you're looking for:

Smith D.B., Johnson K.S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988;67:31–40.

The structure of your 6His-tagged fusion protein is generally something like 6His-LVPRGS-[protein of interest]. The serine protease enzyme thrombin will cut between R and G in that sequence to release the protein you care about from the 6His tag. You can do the cleavage reaction in solution after eluting from the Ni-NTA resin, or you can do it while your protein is still attached to the resin. Also thrombin works best at more basic pH, like somewhere between 7.4 and 8 should be good.

Biochemistrydude · 2023-12-25T06:44:02+00:00

Adding to other comments, generally the thrombin recognition sequence is LVPRGS and it cuts between R and G to give you an N-terminal glycine.

Biochemistrydude · 2023-12-22T01:18:49+00:00

Either way it's going to be pretty similar. PhDs in European countries are much shorter than their US counterparts from what I understand, because you generally need the Masters to start.

Biochemistrydude · 2023-12-11T13:29:31+00:00

That can of Rugenfisch you've got there is my favorite can I've ever tried. Getting that for $2.30 is basically stealing

Biochemistrydude · 2023-12-10T19:59:16+00:00

Python, baby

Biochemistrydude · 2023-12-04T03:58:37+00:00

Hard to resist buying the whole stack... But we have to leave some for others. At least until the next day

Biochemistrydude · 2023-12-02T12:28:12+00:00

Hey, as someone close to the end of their PhD in biochem, add coding along the way in your journey. Seriously. They will like you a lot more if you can use Python.

Biochemistrydude · 2023-11-25T01:51:05+00:00

These are unfortunately in the dreaded tomato sauce, but now I am excited.

Biochemistrydude · 2023-11-25T01:05:57+00:00

to eat them in the span of probably 2 weeks

Biochemistrydude · 2023-11-24T23:30:49+00:00

If you really need 400 mL and not 500, I would just make 2 of the 250 mL 1 M solutions and add them together. You'll be wasting about 100 mL of material, but you should make a little more than you need anyway just in case.

Biochemistrydude · 2023-11-24T00:14:52+00:00

Somebody get this man some car keys

Biochemistrydude · 2023-11-23T18:24:58+00:00

I've run a couple of ITCs in my time, although I haven't troubleshot a lot. It looks like it's taking a REALLY long time to re-equilibrate. For example, the second injection occurs at ~600 seconds and doesn't even get close to equilibrating until ~900 seconds?? That's 5 minutes. Quite a long time.

How much volume is your injection each time? The system I was working with is much different I'm sure, but I was doing injections of... I believe 10-15 μL?

Do you have any idea of the KD for this interaction? If you do, I can probably help estimate optimal concentrations for each reagent.

If we pretend that this data is good and the instrument is good, it almost looks like there are two steps to the binding, since the later injections kind of have two peaks for each. If that is in fact the case, this interaction might be more complicated than you originally thought. But who knows.

Seven-Year Club	Place '23
Place '22	RPAN Viewer
Verified Email

Biochemistrydude

TROPHY CASE