biology_06er

Hi there,
I have to do a cell wall extraction on bacteria soon and my protein of interest is cell wall anchored. My supervisor told me to do TCA precipitation (haven't done before) but I assume it's too isolate the proteins within the cell wall?? He told me I would have three components by the end of it, the soluble, insoluble and something else!!! (the isolated proteins??) arrg I have looked at a protocol for a cell wall extraction. Here is a simplified version below

Wait till OD 0.6
Spin at 5000rpm for 10 mins
Remove Supernatant and resuspend pellet in PBS
Spin at 5000rpm for ten mins
Discard Supernatant and resuspend pellet in 1 mL protoplast buffer. Incubate for 3hours at 37C
spin at 13K for 15mins
store supernatant at -20C

So the supernatant will contain cell wall and the insoluble protoplast component will be left right? So is it the supernatant at the last step i TCA precipitate? if it is, i've looked at a protocol for TCA precipitation and if i followed that, i'd only be left with 2 components. One more thing-there is a note on file (im doing this on behalf of someone not here currently that they used 50mL of supernatant concentrated down to 100uL to detect the protein on a western blot)..How do I get 50mL of supernant? Please help. I'm so confused

Thanks!!
b_06er

Hi there,

I plan to do some flow next week and an one of the steps is to resuspend the bacterial pellet to an OD600 0.5. So say I had a 1.5mL overnight culture and the following morning the OD600 was 1.7. Once I spin down, wash etc etc I read to get to a certain OD (0.5 in my case)...this is the calculation

Divide the OD you want (0.5) by the OD bacteria was at in morning (1.7)=0.5/1.7=0.29=giving you a concentration factor (cf)
Divide the original culture volume (1.5mL) by the cf
1.5/0.29=5.1mL

Ok..so I don't need 5.1mL of culture to subsequently use..say altogether I only need 2mL how do I go about this? So that in the end I have 2mL of bacteria with an OD of 0.5?

Man, I hope I make sense!
Cheers,
b_06er

Hi there,

I tried to make a GFP fusion protein (my gene of interest was fused to the C terminal of GFP) but no expression occured...I tested the expression of the two proteins alone and individually they worked but not when fused together..I tried a linker between the two..still no luck...could it just be that the fusion protein is somewhat toxic to the cell (doesn't seem likely aye..especially if the 2 alones are ok)

cheers,
b_06er

Hi there,

What does it mean when it says "wash twice with PBS" regarding bacteria..does it mean after I spin down my bacteria, I pour off the supernatant and then add PBS and then spin again, pour off s/n add more PBS and spin...

OR DOES IT MEAN

just add PBS ensuring the pellet doesn't break up and then pipette out the PBS and then add more and take it out..?

cheers,
b_06er

Hi there,

I'll be doing FC for the first time next week, and I have a protocol but I'm slightly confused about one thing...after I resuspend my cells in PBS/BSA it tells me to take 200uL of cells and incubate with 1:200 of primary antibody...so does this mean if I have 200uL of cells, I add 1uL of my antibody...and then if I wanted to to another conc of antibody say 1:400 I would add 0.5uL of antibody...I recall being told I would have to do serial dilutions of my primary antibody..but how do I go about doing serial dilutions..if i need 200uL of cells to start with?

Hope I make sense!
cheers,
b_06er