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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
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In Topic: pET14b vector with insert transformed in BL21(DE3) RIPL cells
04 December 2012 - 02:41 AM
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
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
In Topic: pET14b vector with insert transformed in BL21(DE3) RIPL cells
18 November 2012 - 10:40 PM
it is ok with frozen pellets as well
