The coding sequence of CALB1 was cloned into the expression vector pGEX-4T-1 (Clontech Laboratories, Inc.). A PCR was performed using cDNA derived from OVCA429 cells as template. The primers used to clone CALB1 were as follows: Forward, 5′-ATGGCAGAATCCCACCTGCA-3′ and reverse, 5′-CTAGTTATCCCCAGCACAGAG-3′. The PCR was performed using KOD polymerase (Takara Bio, Inc.). The thermocycling conditions of the PCR were as follows: Initial denaturation at 95°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 56°C for 1 min, 72°C for 2 min, with a final extension at 72°C for 10 min. The fusion protein GST-CALB1 was purified as previously described (21 (link)). The whole cell lysate of OVCA429 cells was extracted using a lysis buffer (containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tergitol-type NP-40 and a protease inhibitor cocktail). The protein concentration was determined using the bicinchoninic acid assay. Subsequently, 5 µg GST-CALB1 fusion protein and 500 µg total protein from OVCA429 cells were incubated overnight at 4°C. In total, 50 µl Glutathione Sepharose 4B beads (GE Healthcare) were added to the samples and incubated at 4°C for 1 h to allow the binding between the beads and the GST fusion protein. Following three washes with lysis buffer, the proteins were eluted in Laemmli buffer and analyzed by SDS-PAGE as aforementioned.
Pgex 4t 1
Manufactured by Takara Bio
Sourced in United States
The PGEX-4T-1 is a bacterial expression vector that allows for the expression of recombinant proteins in Escherichia coli. It features a tac promoter for high-level protein expression, an N-terminal glutathione S-transferase (GST) tag for purification, and a thrombin cleavage site for removing the tag after purification.
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Lab products found in correlation
Kod polymerase, by Takara Bio (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Hybond n nylon membrane, by Cytiva (1 mentions)
Magnegst protein purification system, by Promega (1 mentions)
Amersham imager 600, by Cytiva (1 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
Dialysis bag, by Merck Group (1 mentions)
Beyogold gst tag purification resin, by Beyotime (1 mentions)
Pact2, by Takara Bio (1 mentions)
Pcdna3.1a, by Thermo Fisher Scientific (1 mentions)
Human fetal brain cdna library, by Takara Bio (1 mentions)
4 protocols using pgex 4t 1
1
CALB1 Protein Purification and Interactome Analysis
2
DED1 Binding Site Identification
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The Ded1 ORF was cloned into pGEX-4T-1 (Clontech) to express GST-DED1 fusion protein in E. coli strain BL21. The fusion protein was purified using the MagneGST Protein Purification System (Promega). Oligonucleotide probes containing DED1 predicted binding sites from the fl3, sus1, c1, and vp1 promoters were synthesized and labeled with the Pierce Biotin 3′ End DNA Labeling Kit (Genewiz). Labeled single-stranded oligos were annealed to reverse complement unlabeled oligos. Probe sequences are listed in Supplementary Table 1 .
Protein-DNA binding reactions included 50 ng of purified DED1-GST, 6 ng of biotin-labeled annealed oligonucleotides in 20 μL of 10 mM Tris (pH 7.5), 50 mM KCl, 1 mM DTT, 2.5% (v/v) glycerol, 5 mM MgCl2, 50 μg/μL poly(dI-dC), and 0.05% (v/v) NP-40. The reactions were incubated at 25 °C for 20 min, electrophoresed on 6% (w/v) polyacrylamide gels, and transferred to Hybond N+ nylon membranes (Amersham Pharmacia Biotech). Biotin-labeled DNA was detected using a LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific) and imaged with an Amersham Imager 600.
Protein-DNA binding reactions included 50 ng of purified DED1-GST, 6 ng of biotin-labeled annealed oligonucleotides in 20 μL of 10 mM Tris (pH 7.5), 50 mM KCl, 1 mM DTT, 2.5% (v/v) glycerol, 5 mM MgCl2, 50 μg/μL poly(dI-dC), and 0.05% (v/v) NP-40. The reactions were incubated at 25 °C for 20 min, electrophoresed on 6% (w/v) polyacrylamide gels, and transferred to Hybond N+ nylon membranes (Amersham Pharmacia Biotech). Biotin-labeled DNA was detected using a LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific) and imaged with an Amersham Imager 600.
3
Recombinant ClfB Protein Generation
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To generate recombinant ClfB protein, a full-length ClfB DNA fragment was amplified by PCR by using ATCC 25923 strain chromosomal DNA as template and subcloned into expressing vector pGEX-4T-1 (Clontech, Mountain View, CA, USA). PCR primers were as follows: forward 5′-GGT ACC ACA TCA GTA ATA GTA GG-3′, reverse 5′-TCT TTA TGA TCT TGC TTG CGT T-3′. The recombinant GST-tag ClfB protein was produced in BL21 bacteria strain, purified by using BeyoGold™ GST-tag Purification Resin (Beyotime Biotech, Nanjing, China) according to the manufacturer’s protocol, and dialyzed at 4 °C by using a dialysis bag with molecular weight cut-off of 8–14 kDa (Sigma-Aldrich, Saint Louis, MO, USA). The dialyzed protein was stored at −80 °C prior to use.
4
Cloning and Expressing ARL4 Proteins
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cDNA sequences of Arl4A/C/D and its mutants were cloned as previously described (Li et al., 2007 (link)). Briefly, Arl4A was cloned into the mammalian expression vector pSG5 (Stratagene, La Jolla, CA) to generate untagged Arl4A. The cDNA sequence was subcloned into pET32a (Novagen, Madison, WI) for His-tagged Arl4A expressed in Escherichia coli or pBTM116 for LexA-tagged Arl4A expressed in yeast. Full-length Robo1 and srGAP1 cDNA sequences were amplified from a human fetal brain cDNA library (Clontech) using PCR. Robo1 and srGAP1 were cloned into the mammalian expression vectors pCMV-Tag4A and pcDNA3.1A (Invitrogen), the E. coli expression vector pGEX4T-1, or the yeast expression vector pACT2 (Clontech). Replacement of alanine was accomplished using a two-step PCR technique. All constructs were confirmed by DNA sequencing.
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