2xzwei

Hi!

I've overexpressed a protein in both bacteria and human cells (293T). This protein is immunomodulatory and can inhibit TNFalpha secretion of immune cells. So I designed a wildtype and also mutated form that should be expressed by both species.

Now I have this curious findings:
The WT protein from both bacteria and 293T is working well in-vitro (positve effect).
The MUT protein from 293T cells has no effect at all (that's alright as it should serve as negative control).

BUT: The MUT protein from bacteria is also able to abrogate TNFalpha secretion (which should not be the case!).

I've confirmed the sequences of the vectors that have been used for transformation and they were okay. Also the isolated plasmid from bacteria carries the mutation and is fine.
Furthermore the MUT protein from 293T is recognized by ELISA, but NOT the MUT protein from bacteria (although they should be the same). So I'm wondering whether the folding of the MUT protein is different in human 293T and bacterial cells?! Or what explanation would you give for this fact?

I'd be grateful for some advice...
Regards

Hi!
I prepared a 96 well plate with capture AB and over night incubation @ 4°C.
I washed twice and put PBS+10%FCS in each well to block. Unfortunately I cannot continue work today, so is it possible to leave the blocking solution on the wells @ 4°C for 2 days or might this be detrimental? As I already incubated with capture AB I don't want to discard the plate...
What can I do?

Hi!
I've managed to design bacteria that are able to secrete a protein out of the interleukin family. Unfortunately this protein has no HIS-tag etc. for positive selection.
Because the secreted amout of protein is very low (12ng/mL) I have to do a purification/concentration step which I have never done bevor. I did some reading and for the most part people use chromatography to get their protein. Are there any other (simpler and low-price) methods that one can do in the lab?
Thanks in advance

Hi there!

Currently I'm struggling with the isolation of a plasmid from Enterococcus faecalis (gram positive bacteria). In a first run I tried it by following the standard protocol for plasmid extraction (Qiagen kit) but I didn't yield any plasmid. So I read about the addition of Lysostaphin which should crack the murein layer (0.5mg/mL 10µL per 250µL suspension) and incubated it for 45min @37°C. But this didn't help, too

Now I tried the addition of Lysozyme (in Tris-HCl buffer pH 8) to the suspension buffer (100µL of 10mg/mL Lysozyme stock) and incubated 30min @37°C. But all I got was some smear on my agarose gel and no plasmid...

Does anyone of you have further suggestions how to successfully isolate plasmid from gram positive bacteria (esp. E. faecalis)? I read about methods with heat-lysing (heating to 80°C for a while) but I couldn't find any protocol for that...

Thanks in advance for your help!

PS: Yes the plasmid is inside, otherwise it wouldn't grow on my antibiotic agar.

Hi!

I've been trying for months now to insert a 122bp ds oligo into a 8000bp vector but it just doesn't work. I tried vector:insert ratios from 10:1 to 1:5, different ligases/buffers and also changed the time for ligation (2h - over night at RT, 4°C and 16°C). But nothing worked so far. So now I think there might be something wrong with my oligo design.

The setup was as follows:

2 single strand oligos, annealing: 10 nmol each, 5min 99°C, then let cool down to RT (4h). I used annealing buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA).




The oligo contains compatible overhangs for BamHI and Pst1 but as you can see both strands were designed 5' to 3' (manufacturers data sheet) so is that the reason why the ligation cannot take place?
The oligos are not phosphorylated but the cut vector should have PO4 groups from restriction digest. Can I do something to use both oligos at the same time or do I have to design at least one with reverse direction (3' - 5')?

Anyway, is there any method to check whether the annealing has worked or not - how can I distuinguish if I have single strand or double strand oligo after annealing?

Thanks in advance!