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IPTGInstitute of Petroleum Technology Gandhinagar (India)
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After induction by 1 mm IPTG at 37AdegC for 4 h, the Sf200 proteins were found in the inclusion bodies in cell lysate of E.
The C426 avimer recombinant protein was expressed under IPTG induction and detected using SDS-PAGE (according to band abundance the concentration of protein is predicted to be 5 [mu]g/[mu]l) and western blotting.
coli BL21 (DE3) and expressed under the influence of IPTG. The resultant recombinant protein was run on SDS-PAGE gel that revealed successful expression of the protein indicated by the expected size (Figure 1(b)) and was confirmed by western blotting using anti-his antibody (Figure 1(a)).
Expression host (E.coli strain BL21) containing plasmid encoding the recombinant protein was cultured overnight in different temperatures and was induced by IPTG until the recombinant protein expressed.
The isopropyl-AY-D-thiogalactopyranoside (IPTG) is commonly used inducer to regulate the expression of target proteins under the influence this promoter in BL21 (DE3) strain.
After the cell OD600 reached 0.4-0.6, IPTG was added to the final concentration of 1mM.
When the O[D.sub.600] of the culture reached 0.8, the promoter of the recombinant vector was induced by the addition of IPTG to the final concentration of 1 mM.
coli culture harbouring pQE (OD600 of 0.6 was induced with 1mM IPTG to express the recombinant 18.5 protein and grown again for 4 hr at 37[degrees]C in a shaker incubator at 180 rpm.
Later, the transformed cell culture was plated on LB medium supplemented (100 g/mL) ampicillin, X-gal 5-bromo-4- chloro-3-indolyl-AY-D-galactosidase (40 ng/mL) and IPTG isopropyl-AY-Dthiogalactopy-ranoside (0.5 mM) and incubated over night at 37C.
IPTG induction, expression analysis, and GST proteins immobilization on glutathione-Sepharose 4B beads were performed as previously described [22].
When the culture had grown to an [OD.sub.600] of 0.6-0.8, the expression of recombinant FSHb was induced by adding isopropyl-[beta]-D-thiogalactopy-ranoside (IPTG) at 0.1 M final concentration, and incubating for 1, 2, 3 and 4 h at 37[degrees]C.
Transformed cells were plated on 200 mL of selective medium (Luria-Bertani medium) in 24- by 24-cm plates (Genetix) with 12.5 [micro]g [mL.sup.-1] chloramphenicol, 0.55 mM isopropyl-beta-D-thiogalactopyranoside (IPTG), and 80 [micro]g [mL.sup.-1] X-Gal.