pET32(a)+-BLS

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The BLS fragments sub-cloned in pET32 (a) + vector (Novagen, USA), and subsequently was transformed into E.coli BL21 (DE3) expression host strain (Novagen, USA). The harboring transformed bacteria was spread on LB agar containing 50 µg/mL ampicillin (Sigma, Germany) cultivation and incubated at 37 °C overnight.	To analyze the expression of recombinant BLS both the supernatant and the pellet were evaluated on SDS-PAGE 10% (upper gel was 8% and lower gel was 10%).

Protocol tips
The BLS fragments sub-cloned in pET32 (a) + vector (Novagen, USA), and subsequently was transformed into E.coli BL21 (DE3) expression host strain (Novagen, USA). The harboring transformed bacteria was spread on LB agar containing 50 µg/mL ampicillin (Sigma, Germany) cultivation and incubated at 37 °C overnight.
Downstream tips
To analyze the expression of recombinant BLS both the supernatant and the pellet were evaluated on SDS-PAGE 10% (upper gel was 8% and lower gel was 10%).

Publication protocol

For the expression, BLS open reading frame was digested by NcoI and EcoRI restriction enzymes. The excised fragments were purified from agarose gel using commercial Kit (BioRon, Germany). The BLS fragments sub-cloned in pET32 (a) + vector (Novagen, USA), and subsequently was transformed into E.coli BL21 (DE3) expression host strain (Novagen, USA). The harboring transformed bacteria was spread on LB agar containing 50 µg/mL ampicillin (Sigma, Germany) cultivation and incubated at 37 °C overnight. One positive BL21 clone was selected using colony PCR with T7 universal primers. The presence of BLS coding sequence was confirmed by restriction endonuclease analysis. The positive colonies were cultured in selective LB medium at OD = ~ 0.6. Moreover, IPTG (isopropyl βDthiogalactoside) was added at the final concentration of 0.1mM to induce the expression of the BLS protein at 37°C. Harvest cells were suspended and lysed using lysis buffer and sonication (Yousefi et al., 2016). Cell lysate, was centrifuged at 9000 g for 15 min at 4 °C to separate the supernatant containing soluble materials from the pellet. To analyze the expression of recombinant BLS both the supernatant and the pellet were evaluated on SDS-PAGE 10% (upper gel was 8% and lower gel was 10%). The expressed protein was purified using Ni-agarose chromatography (Thermo, USA) from the insoluble phase of lysate using guanidine hydrochloride 6 M to dissolve the pellet. The quality and identity of the recombinant BLS protein was analyzed using SDS-PAGE (10%) and western blotting assay, respectively. For western blotting, the SDS-PAGE gels were electroblotted onto nitrocellulose. Afterwards, the blotted nitrocellulose was blocked with skim milk for 3 h. The membranes were washed 3 times and then Anti Poly-Histidine-HRP (Sigma, 1:2000 diluted in BSA 1%) was added. For visualization, the incubation was at room temperature, and membranes were washed with diaminobenzidine (DAB) as chromogen an hour later. Finally, the quantity of the recombinant protein was estimated using Bradford assay (Yousefi et al., 2016). The purified recombinant BLS protein was stored at -20 °C for further studies.

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