ascacioc

Sorry for taking me ages to reply. I have been on vacation.

beads experiment: take the pellet from 50 mL of culture, resuspend in 5 mL of buffer, sonicate, spin at highest centrifugation speed your table centrifuge allows (at 4oC) and then incubate 2 mL of supernatant for ~2h with 20 uL of affinity beads (equilibrated in the proper buffer). Then wash the beads 3-5 times with the buffer while centrifugating at low speed for 2 min in between washes. Load this on the SDS-PAGE.

Interaction partners: well, it might not work as your professor thinks. You need a very stable interaction and if you have a transient interaction as in regulatory pathways, these are not stable complexes (trust me, I also have the nightmare of transient complexes in my PhD thesis). Moreover, even if you have a stable interaction, if you have more than 150 mM salt (NaCl or KCl) in the binding buffer, this complex might disassemble. Furthermore, if you have to elute the protein from the column, you need to add imidazole, which is a salt and will increase the total amount of salt in the buffer so your interaction partner will dissociate before your his-tagged protein.

Your frozen pellets are good forever...ok not forever, but until the end of your project as long as it does not last 10 years. So, yes you can use the ones you have in the freezer.

Andreea

I am also confused myself. Is that a 15% gel? I guess it must be if you want to see your protein of 16 kDa Anyhow, what I can tell you is that sometimes in the uninduced fractions you could also see your protein when you have leaky expression which can happen as T7 from pET system is a very strong promoter and it is very difficult to inhibit before expression is induced. I would do this experiment with the beads that I have suggested above, like this you can observe whether the enriched fraction contains a protein around your size. I would guess that the pink band did not separate enough and that you have from bottom to top: 6, 15 and so on and the top marker bands that run all together contain the pink band as well. I would not continue until I see the enriched band on the gel from the beads experiment. However, you could also try direct large scale expression. Most people around me are lucky enough for it to work from the first trial

Andreea

BTW: when you harvest your cells for purification you can also freeze the pellets before you start the purification which will take the entire day

it is ok with frozen pellets as well

For the first part: I usually measure undiluted, but it works like you too; actually, if you are trying to get OD600 = 1 then you should dilute. I induce myself at 0.6
For the second part: you can keep your pellets also at -20 (in case you need space in your -80; I surely always have this problem)
I always resuspend the pellet in 5 mL of buffer and take 100 uL of this + 50 uL 3X loading buffer (load only 3 uL on a gel because it is very concentrated); sonicate these 5 mL and spin down for 30 min at the max speed of the centrifuge I have at 4oC; take 100 from supernatant and treat as above; from the pellet, resuspend it in 2 mL of water and take 100 uL and treat as above; the rest of the 5 mL of the soluble fraction I incubate with 20 uL affinity beads for my tag on rotation in the cold room for 1-2 h and then wash 3-5 times and then boil the beads in sample buffer and load 10 uL of this on a gel.

Andreea

1 - you have a promoter before your gene; the RNA polymerase recognizes the promoter and then it goes in the direction of the gene; different gene directions means that the gene is either on the upper or bottom strand; when the RNA polymerase binds to the promoter, it chooses which one is the coding strand of the two possibilities
2 - Both Tet and Amp genes have their own promoters (see above for direction) and terminators. Tet stops before the origin since there is a terminator before the origin starts; hence, there is no collision

Andreea

In your case, I would use the 15% SDS-PAGE gels as described in Sambrook and Maniatis molecular cloning: http://www.amazon.de...l/dp/0879695773

Loading buffer I prepare and aliquot in 1 mL Eppis at -20; the rest I keep at RT. I never prepare gels ahead of time and then store them. People do that and keep cast gels in the fridge but over time the H+ gradient between the stacking and the resolving (which are usually at different pHs) will diffuse and the resolution of the gels will be sub-optimal.

Andreea

I usually spin it down and discard the media and then freeze the pellets at -20 until I have time to analyze them.

Andreea

Step 4: add only 1 mL in 100 mL: usually it is 1 in 100 dilution. The rest is beyond perfect. I know that you said that you read the materials from me, but now you even proved it

Andreea

You got it all right with the culture, as I explained.

Do not worry about how different people do the same thing in many ways. Choose whatever works for you, and stick to it.
I do 50-100 mL of preculture. Depends on what flask I have available I usually make 1/10 of the volume of the flask i.e. for 500 mL flask I do 50 mL preculture.
Usually, I measure OD600 in a cuvette not with the microplate reader (too complicated and takes too long); I just take 1 mL culture, put in a cuvette and stick it in the spectrophotomer. Moreover, measure the culture every 1 hour or even 30 min. Keep in mind that the duplication time of BL21 derived bacteria is 20 min (around, depends on what protein you are expressing) so if you are measuring every 2 hours you get 0.1 and next point is >6.

I usually do 6 L of main induced culture but you need the flasks and shaker capacity for this kind of expression and BTW, I do X-ray crystallography so I need tons of my protein Another important aspect: I use 1/5 of the volume of the flask for expression i.e. in a 5 L flask, I put 1 L of media. This is needed for good aeration.

And another thing: before I waste my time with 6 L of culture, I make an expression test in 100-200 mL of culture from which I take several time points 50 mL and harvest the cells, resuspend in 5 mL, brake them with a sonicator, and run a gel with pellet, soluble protein (supernatant of the disrupted cells) and I also incubate for 1-2 hours on rotation 2 mL of supernatant = soluble protein solution with 20 uL of the affinity beads i.e. Ni-sepharose for His-tagged proteins to get a sample of enriched protein (for the SDS-PAGE, I wash these beads 3 times at low centrifugation speed and then boil them in loading buffer)

Andreea

yeah... but you don't know who took them out and in after 1-2 hours in the weekend without saying anything (I have seen similar things happening). I would inoculate in a liquid culture directly from the stock and see what happens. On the other hand, I would never trust that culture if I do not see single bacterial colony on the plate. On the other hand, if you still have the DNA, what would stop you from getting a new transformation? It might be a bit too much work, but better do it again than forever wonder whether what you are working with is the real thing and about where the problem is. Just my two obsessive compulsive-paranoid cents...

Andreea

No-no: I said: put both on agar plate and the overnight preculture, but when you make your big culture, put only ampicilin: aka the 1-6 L of LB which you induce with IPTG should contain only one antibiotic because 2 of them will be too stressful.

Andreea

In addition: also the preculture should contain both antibiotics. However for the main expression I would use only ampicillin because 2 antibiotics at once during expression make to much stress on the cells.

Andreea

So that's where you were Best of luck. Keeping my fingers crossed... and if they don't take you: Europe is always looking for good scientists Malaysia's loss is our win

as above At least when I was a student I was google-ing the info to patch together in an answer not expecting other people to solve my homework