I’m working on recombinant protein expression.
My bacteria lysis protocol:
The grown cells (Bl21) were harvested by centrifugation at 5000g for 20 min at 4 ◦C, resuspended in 5mL of PBS buffer, pH 7/4. Then cells were incubated with lysozyme (0.3 mg/mL). After that, bacteria were disrupted by 16 cycles of sonication of 30 s each(30s pulse,30s stop total time: 8min). Then, the lysed cells were centrifuged at 12,000 rpm for 15 min. at 4 ◦C.
The supernatant and Precipitation dissolved in ureatasted by SDS-PAGE 7/5% But I have seen NO BAND for the supernatant.
Protein molecular weight: 116kDa
If you have any experience can you help me?
My guess is either the sonication did not work at all and the cells were not broken, or the excessive amount of sonication overheated the sample so much that all the protein precipitated.
In future attempts, you should monitor the progress of the cell breakage after each cycle of sonication. One way to do this is to look at the cells under the microscope to see if they are intact. Another way is to measure the protein concentration in the supernatant.
Hi,
You might not have your recombinant protein being expressed in this sample. You can check by taking a small amount (~50 uL) of your induced culture and lysing the cells with SDS-PAGE and running the entire lysate on the gel along with an uninduced control. If your recombinant protein has an affinity tag, you can also do a quick purification and run it on a gel.
Good luck!
Hi, I had a very similar problem. I used homogenization instead of sonication and LB instead of TB. You can also try, remember that LB expression procedure is different to TB's.
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