FAQ
The last number refers to the reading frame of the vector. pGEX-6P3 has an additional base in the multiple cloning site compared with pGEX-6P2, which in turn has an additional base compared with pGEX-6P1. Multiple reading frames enable you to select the appropriate vector for your application to avoid frameshift.
The letter refers to the protease recognition sequence for that vector:
T, Thrombin protease;
X, Factor Xa;
P, PreScission Protease
The table includes additional details for the three proteases.
Protease | Molecular weight | Pack size | Capacity | Application |
---|---|---|---|---|
PreScission Protease |
46 000 | 500 units | One unit cleaves > 90% of 100 µg of a test GST-tagged protein when incubated in 1 mM EDTA, 1 mM DTT, 150 mM NaCl, and 50 mM Tris-HCl (pH 7.0) at 5°C for 16 h. | For specific, low-temperature cleavage between Gln and Gly residues in the sequence Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro. A tagged protein consisting of human rhinovirus protease and GST. |
Can be used for tag cleavage when the PreScission Protease recognition sequence occurs between the tag sequence and the protein of interest. (e.g., the GST tag from proteins expressed using the pGEX-6P vector). | ||||
Bovine thrombin |
37 000 | 500 units | One unit cleaves > 90% of 100 µg of a test GST-tagged protein when incubated in 1× PBS at 22°C for 16 h. | Serine protease for specific cleavage at the recognition sequence for thrombin. Can be used for tag cleavage when the thrombin recognition sequence occurs between the tag sequence and the protein of interest (e.g., the GST tag from proteins expressed using the pGEX-T vectors). |
Bovine Factor Xa |
48 000 | 400 units | One unit cleaves > 90% of 100 µg of a test GST-tagged protein when incubated in 1 mM CaCl2, 100 mM NaCl, and 50 mM Tris-HCl (pH 8.0) at 22°C for 16 h. |
Serine protease for specific cleavage following the tetrapeptide Ile-Glu-Gly-Arg. Can be used for tag cleavage when the Factor Xa recognition sequence occurs between the tag sequence and the protein of interest (e.g., the GST tag from proteins expressed using pGEX-X vectors. |
A range of Gluthatione Sepharose 4B prepacked columns and bulk media are available to purify GST fusion proteins.
Sample volume | Prepacked column |
---|---|
< 600 µl | GST SpinTrap spin column (28-9523-59) or GST MultiTrap 4B 96-well plates (28-4055-00) |
600 µl–10 ml | GST GraviTrap gravity flow column (28-9523-60) |
>10ml | LabMate PD-10 Buffer Reservoir (18-3216-03) together with GST GraviTrap (28-9523-60) |
All formats described can be used for preparation of samples in parallel. In addition, GST HiTrap and GST HiPrep columns are available for purification using a chromatography system such as an ÄKTA system. Alternatively, Gluthatione Sepharose 4B bulk medium is available from 10 ml up to 300 ml. A GST Bulk Kit is also available, combining 10 ml Gluthatione Sepharose 4B bulk medium with the required buffers. For simplified buffer preparation use the GST Buffer Kit.
A280, UV absorbance at specified wavelength (e.g., 280 nm)
ABTS™, 2',2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt
AC, affinity chromatography
AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride
BCA™, bicinchoninic acid
CDNB, 1-chloro-2,4-dinitrobenzene
CV, column volume
DMSO, dimethylsulfoxide
DNase, deoxyribonuclease
DTT, dithiothreitol
ELISA, enzyme-linked immunosorbent assay
FF, Fast Flow
Gua-HCl, guanidine-HCl
GF, gel filtration
GST, glutathione S-transferase
HP, High Performance
HRP, horseradish peroxidase
IPTG, isopropyl β-D-thiogalactoside
LMW, low molecular weight
MCS, multiple cloning site
MWCO, molecular weight cutoff
MPa megaPascal
Mr, relative molecular weight
PBS, phosphate-buffered saline
pI, isoelectric point, which is the pH at which a protein has zero net surface charge
psi, pounds per square inch
PMSF, phenylmethylsulfonyl fluoride
PVDF, polyvinylidene fluoride
R, recombinant, as in rGST and rBCA
RNase, ribonuclease
SDS, sodium dodecyl sulfate
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide electrophoresis
Vector | Protease for cleavage |
---|---|
pGEX-6P-1, pGEX-6P-2, pGEX-6P-3 | PreScission Protease |
pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-1λT, pGEX-2T, pGEX-2TK | Thrombin |
pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pGEX-3X | Factor Xa |