I produced recombinant βA domain of β-Integrin protein from shrimp using recombinant plasmid pGEX4T1- βA. The plasmid was transformed into E. col BL21 Star. The cells were induced by 0.5 mM IPTG. The recombinant βA domain was expressed after induced by 0.5 mM IPTG. The cells were centrifuged and soluble was discarded. The cell pellets were lysed by 30 ml lysis buffer (PBS pH 7.4 containing 0.1% Triton X-100 1 mM PMSF). The protein was analyzed by 12.5% SDS-PAGE. After analyzed, i found that the recombinant βA is missing. Could you please suggest me what should i do to solving problems?
Note: recombinant βA domain was tagged by GST. The recombinant protein have a size 57 kDa.
Pic detail: Lane N: non-induced Lane 1-4: induced Lane B: after added lysis buffer Lane: I: inclusion body Lane S: Soluble
How did samples 1 to 4 are obtained? How did you solubilize inclusion bodies?
Condition of sonicated
VCX 750 750 Watt Ultrasonic Processors
5 sec On / 5 sec Off pulser for 30 min
25% Amp
Dear, Omar Gonzalez-Ortega
I culture recombinant bacteria in 250 ml LB containing 100 ug/ml Amp and 34 ug/ml CamR.
1 ml of sample 1- 4 was kept after induced at 1, 2, 3, and 4 hours. The samples were centrifuged to keep cell pellets. The cell pellets were dissolve by 20 ul pbs buffer pH 7.4 and mix with 6x loading buffer. After mix the samples were boiled at 100 °C for 15 min and take 10 ul to analyze by SDS-PAGE.
Inclusion body and soluble are protein from cell pellets of 250 ml after sonication.
Inclusion body was dissolve by 30 ml PBS pH 7.4 containing 5 mM EDTA and 2 M Urea.
Both sample (20 ul) were mixed with 6x loading buffer. After mix the samples were boiled at 100 °C for 15 min and take 10 ul to analyze by SDS-PAGE.
Sincerely,
Prawit
2 M urea is not enough to dissolve inclusion bodies, increase the conc to 8 M and allow dissolution for 24 h in the fridge.
Are you sure it is completely lysed? Use some other method of lysis also. Please check the soluble also.
Sonication is not the gentlest method of homogenisation. If you have access to a french press, try that.
I am not sure about the samples in your electrophoresis. Do lanes 1-4 represent bacteria before or after homogenisation? And what about "after adding lysis buffer"?
It would be a good idea to analyse by electrophoresis whole bacteria in growth medium, in lysis buffer, directly after lysis, and then supernatant and pellet after centrifugation. Make sure that the amount added to each lane corresponds to the same volume of culture, so that the results can be directly compared. It would also be useful to make a balance sheet for each step: how much protein did you put in and how much did you get out (both concentration and amount). If you have an assay to measure the concentration of your protein (enzymatic activity, immunoreactivity ...) then also keep book about that. You can then calculate the specific activity (i.e., purity), which should increase from step to step.
PMSF alone may not suffice to prevent proteolysis, you may need additional protease inhibitors. However, that is best judged after you have figured out in which step the loss occured.
Thank you very much for all your suggestions. Now i find my protein by using 8 M Urea to dissolve.
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