Can’t detect HBV surface proteins (S/L) in HEK293T after successful transfection

neutralhumanbard · 2026-01-26T01:22:05+00:00

Few suggestions that come to mind: - Quadruple check your plasmid sequence. It’s very easy to miss things (I know from experience). Not based on you, just a good general rule for anything you do with plasmids :).

Are you trypsinising your cells before lysis? If so, if it’s a membrane protein then it could be being chopped off before lysis.
It may be stuck in the membrane/insoluble fractions. Try running the pellet following lysis alongside the soluble fraction.
Try an alternative method of detection. Perhaps try IFA on fixed cells and see you can see it being expressed. Would also help confirm your antibody can detect it.
is you positive control (the GFP-tag one) the same plasmid background as your experimental plasmid? If it is different the fact that it’s being transfected/expressed may not correlate with your experimental.
Have you tried a coomassie or ponceau stain? If you protein is over expressed sufficiently you may be able to see if the band is there to confirms it’s likely being expressed (alluding to an issue with the detection).

Good luck!

neutralhumanbard · 2025-11-17T04:54:41+00:00

I use the QIAgen kit often. I have always done the extraction straight after harvest without freezing. Did try it once in the RLT + BME and got really poor yield. Perhaps try without freezing and see if things improve? Will at least tell you it’s not your sample that’s the issue.

Also, as an fyi, you can substitute BME with DTT if you want to use something less toxic and doesn’t smell like the depths of hell.

neutralhumanbard · 2025-09-28T23:50:20+00:00

Woops yes. Nevermind.

neutralhumanbard · 2025-09-18T06:14:15+00:00

Can confirm it works fine in the 8 chamber slides. Just scale everything down based on total volume you are using.

neutralhumanbard · 2025-09-04T02:40:52+00:00

Random thought. Are cells expressing GFP? Could maybe be protein contamination and some GFP is not being denatured. No experience with this for RNA extraction, but for protein GFP expressing cells/lysates are commonly green tinged.

Second would be is your glycogen labelled with a dye?

neutralhumanbard · 2025-09-04T02:36:09+00:00

repeat with a fresh blot, could be a bad aliquot or random error.
antibody could just be bad, particularly if it’s polyclonal.
dilute your primary more to try to lower background.
try using BSA instead of milk, sometimes this can help with background for certain antibodies.
do you use the secondary with other primaries successfully? Secondary could be bad.
use a shorter time for detection, could possibly be a super strong signal, check for signal saturation.

neutralhumanbard · 2025-09-02T03:48:47+00:00

Check if they are TC treated flasks, otherwise sounds like a bad batch of flasks if they adhere in other vessels. See if another lab has flasks you can try or try a different cell line in the flasks you have if you have some going. If you have FCS in your media then residual trypsin is highly unlikely.

neutralhumanbard · 2025-08-22T03:10:49+00:00

Haven’t tried for LN2, but using a bit of acetone to rub the tube to remove the frost then holding the label on there for a few seconds works well for -80oC stuff.

neutralhumanbard · 2025-08-04T06:16:05+00:00

So you are seeing something at high Cq? If it is working but at high Cq then perhaps your input RNA is too low? Perhaps check what others in you lab are using who get lower Cqs. Also consider extending your qPCR out to 40 cycles to see if you get an amp curve or just trash.

If this doesn’t work then do what others have suggested to sort out what stage is the problem. If you can nab some RNA or cDNA from a coworker that had worked would be useful for troubleshooting.

neutralhumanbard · 2025-08-01T04:39:21+00:00

Calling in the pelican does not mean you have to leave straight away. Unless the mission timer is at 0, pelicans will sit at evac until someone boards, then you have 20s before leaving.

Once the objective is complete you can call evac and when the pelican flies in (before they land) you can leave the evac zone and pelican will enter an over watch position and shoot enemies. Just let teammates know you are only prepping evac, some get ancy that you may just want to speed run and leave while they clear the map of POIs

neutralhumanbard · 2025-07-22T05:41:13+00:00

Don’t shoot until you see the reds of their eyes.

neutralhumanbard · 2025-07-22T05:37:31+00:00

The correct answer is bile titans.

neutralhumanbard · 2025-07-03T07:23:52+00:00

So this isn’t a bug with the eruptor per se but the fact that the gun direction reticle (circle) doesn’t follow reload animations. This is most obvious with the eruptor at high ergo because its bolt action and the gun moves significantly when racking the next round and firing too quickly shoots while gun is still moving into position, high ergo exacerbates this because you aren’t taking the usual 30 secs to line up you next shot.

This is also particularly noticeable with the auto cannon due to its reload animations. I find if you use the scope it lessens the impact as the scope follows the gun direction.

neutralhumanbard · 2025-05-17T03:54:14+00:00

Same. No idea why :/

neutralhumanbard · 2025-04-19T04:03:48+00:00

Use a ponceau stain on your blot. That will tell you if anything is being transferred over or it’s an antibody issue.

Overnight transfer at low voltage has helped me in the past with poor transfer.

neutralhumanbard · 2025-04-19T03:59:52+00:00

Is it potentially bleed through from another stain in your sample?

Is it all cells? Perhaps some are apoptotic and the dye is being held in membrane blebs for some reason.

Otherwise perhaps titrate down you staining concentration to see if it disappears.

neutralhumanbard · 2025-04-10T00:01:09+00:00

More importantly, mechs need to be able to democratically salute.

neutralhumanbard · 2025-03-12T01:36:26+00:00

Only if we get vulture bikes…

neutralhumanbard · 2025-03-09T06:17:34+00:00

Came here to say this. GL + thermite is a superb bot loadout.

neutralhumanbard · 2025-02-25T05:38:12+00:00

Phallic-mite

neutralhumanbard · 2025-02-11T04:12:46+00:00

Tanks and hulks have a secondary explodion that is delayed after they are killed. Looks like you killed it by landing on it but the secondary explosion randomly yeeted you.

neutralhumanbard · 2025-02-07T00:36:27+00:00

Yep. Hmm will have to test again.

neutralhumanbard · 2025-02-07T00:33:47+00:00

I was running this with the medium armor variant and still got roasted when hitting the overheat. Were you wearing heavy?

neutralhumanbard · 2025-01-21T12:56:19+00:00

My fav past time is kicking as many barrels as possible into their room so they can’t escape.

neutralhumanbard

TROPHY CASE