The scFv antibody, BG4, was purified using the expression vector pSANG10-3F-BG4 (Addgene plasmid no. 55756) according to the previous study (Hänsel-Hertsch et al. 2018 (link)). To get BG4-EGFP antibody, the EGFP coding sequence was amplified from the EGFP-Tpr (Addgene #35024) and subcloned into pSANG10-3F-BG4 to generate the BG4-EGFP expression plasmid, which was chemically transformed into BL21 (DE3) competent cells. Expression of BG4-EGFP was induced by 0.2 mM IPTG for 14 h at 16°C, 200 rpm. BG4-EGFP protein was then purified with the Ni NTA beads 6FF (Smart-Lifesciences). See Supplemental Methods for further details.
Ni nta beads 6ff
Manufactured by Smart-Lifesciences
Sourced in China
Ni NTA Beads 6FF are agarose-based affinity chromatography beads pre-charged with nickel ions. They are designed for the purification of recombinant proteins containing a histidine-tag sequence.
Automatically generated - may contain errors
Lab products found in correlation
Psang10 3f bg4, by Addgene (1 mentions)
Ultrasonic cell disruptor, by Ningbo Scientz Biotechnology (1 mentions)
Ethylenediaminetetraacetic acid, by Beyotime (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonsera (1 mentions)
Ni nta sensors, by Molecular Devices (1 mentions)
Gapdh af7021, by Affinity Biosciences (1 mentions)
P38mapk d13e1, by Cell Signaling Technology (1 mentions)
Triethylammonium bicarbonate teab, by Merck Group (1 mentions)
Tmt kit, by Thermo Fisher Scientific (1 mentions)
Endofree plasmid midi kit, by CWBIO (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Fastpure gel dna extraction mini kit, by Vazyme (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Trypsin edta, by Beyotime (1 mentions)
T7 express lysy iq competent e coli cells, by New England Biolabs (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Rproteina beads 4ff, by Smart-Lifesciences (1 mentions)
7 protocols using ni nta beads 6ff
1
Purification of BG4-EGFP Antibody
2
Recombinant Protein Expression and Purification
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All constructs were transformed into E. coli DH5α cells and selected for with ampicillin (100 µg/mL). Recombinant gene integration was verified by restriction enzyme digestion and DNA sequencing analysis. Selected positive transformants were inoculated into 200 mL LB (liquid Luria broth) containing 200 µL ampicillin (100 µg/mL) and incubated at 30 ℃ with agitation. Then, the incubation temperature was rapidly increased to 42 ℃ in a water bath incubator shaker when the cultures reached the mid-log phase of growth (OD550 = 0.5–0.6). The time point for induction was determined, and the best induction time was 6 h. For purification, the bacterial cells were harvested by centrifugation at 6000 rpm for 15 min at 4 °C and washed three times with PBS (phosphate-buffered saline, pH 7.2). The bacterial pellet was resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and sonicated for 25 min on ice using an ultrasonic cell disruptor (Scientz, Ningbo, China). The inclusion bodies were separated by centrifugation at 10,000 rpm for 20 min at 4 °C, and the inclusion bodies were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 6 M guanidine hydrochloric acid, pH 8.0). Ni NTA Beads 6FF (Smart-Lifesciences, Changzhou, China) was used for protein purification according to the manufacturer’s instructions.
3
Activation of p38 MAPK Pathway
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Reagents: Dulbecco's modified eagle's medium (DMEM) (Sigma-Aldrich, MO, USA); fetal bovine serum (FBS) (Lonsera, UY, South America); p38 mitogen activated protein kinase (p38 MAPK) (D13E1) (8690, Cell Signaling Technology, MA, USA); phospho-p38 MAPK (P-p38 MAPK) (Thr180/Tyr182) (4631, Cell Signaling Technology, MA, USA); GAPDH (AF7021, Affinity Biosciences, Beijing, China); horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (S0001, Affinity Biosciences, Beijing, China); phosSTOP phosphatase inhibitor (Roche, Basel, Switzerland); GC-rich PCR master mix (Sangon Biotech, Shanghai, China); acetonitrile (Fisher Chemical, Fairlawn, USA); triethylammonium bicarbonate (TEAB) (Sigma-Aldrich, MO, USA); Escherichia coli BL21(DE3) competent cells (Solarbio, Beijing, China); Ni NTA Beads 6FF (Smart-Lifesciences, Jiangsu, China); BCA protein assay kit (Beyotime, Shanghai, China); endofree plasmid midi kit (Cwbiotech, Beijing, China); fastpure gel DNA extraction mini kit (Vazyme Biotech, Jiangsu, China); Ni-NTA sensors (ForteBio, CA, USA); tandem mass tagging (TMT) Kit (Thermo, Waltham, USA). Ethylene diamine tetraacetic acid (EDTA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypsin-EDTA (TE) were obtained from Beyotime Technology (Shanghai, China).
4
Detecting HER2-NB2 Protein Interaction
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The interaction of HER2 ECD protein and NB2 was detected by pull-down. Ni NTA Beads 6FF (SA005010, Smart-Lifesciences, China) of 30 μl were added to two EP tubes and washed three times with 1 ml binding buffer for 5 min at 4°C, 5,000 rpm. One was treated with 1 ml pET-28a-HER2 (ECD) supernatant, 1 mM PMSF, and 20 μl NB2 protein, and the other was treated the same, but with NB2 protein overnight at 4°C. The beads were centrifuged (5,000 rpm, 5 min, 4 °C), and washed three times with 1 ml wash buffer each by centrifuged (5,000 rpm, 5 min, 4°C). The beads were treated as above to become the protein samples for analysis.
5
Recombinant Protein Purification Protocol
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The pGEX-GST-His6-HBD and pGEX-GST-His6-2×HBD plasmids were individually transformed into the T7 Express lysY/Iq competent E. coli cells (NEB, C3013). The transformed colonies were picked and cultured in the 2×YT medium containing ampicillin (100 μg/ml) at 200 rpm in a 37°C shaker. Protein expression was induced by 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG) when the optical density at 600 nm (OD600) reached 0.6, and the culture was grown for an additional 5 hours at 200 rpm. Bacterial cell pellets were collected and lysed in 40 ml of HEX buffer [20 mM Hepes-NaOH (pH 7.5), 0.8 M NaCl, 10% glycerol, and 0.2% Triton X-100] supplemented with 1× cOmplete, EDTA-free protease inhibitor cocktails (Roche, 04693132001). Homogenization of lysates was performed with a high-pressure homogenizer at 5.5 MPa for 5 min. The supernatant was collected by centrifuging at 12,000 rpm and 4°C for 30 min. The GST-His6-HBD and GST-His6-2×HBD proteins were purified with the Ni-NTA beads 6FF (Smart Life Sciences) and eluted with the HEX buffer containing 250 mM imidazole. The eluted proteins were dialyzed against the HEX buffer and were concentrated with an Amicon Ultra-15 centrifugal filter unit (Millipore, UFC901008; 10-kDa cutoff). The GST-His6-HBD and GST-His6-2×HBD proteins were further analyzed by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining.
6
Antibody and Affinity Reagents for Protein Detection
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The following antibodies were purchased: anti-Myc antibody (GNI, GNI4410-MC, 1:4000); anti-Flag antibody (GNI, GNI4410-FG, 1:4000); anti-H + -ATPase antibody (PHYTOAB, PHY2285A, 1:2000); anti-Rbcl antibody (Agrisera, AS03037, 1:4000); anti-H3 antibody (Abmart, P30266M, 1:3000); anti-β-Tubulin antibody (Abmart, M30109M, 1:4000); anti-GFP antibody (Abmart, M20004, 1:3000); anti-PIF4 antibody (Abiocode, R2534-4, 1:2000); anti-GST antibody (Abmart, M20007, 1:4000); anti-His antibody (GNI, GNI4110-HS, 1:4000); Alexa Fluor 488-labeled Goat Anti-Mouse IgG antibodies (Beyotime, A0428, 1:200); Glutathione Resin (GenScript, L00206); anti-GFP Affinity beads (Smart-Lifesciences, SA070001); rProtein A Beads 4FF (Smart-Lifesciences, SA015005); Ni NTA Beads 6FF (Smart-Lifesciences, SA005025).
7
Purification and Characterization of CpxR Protein
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The cpxR gene CDS was amplified with the primers cpxRF1 and cpxRR699 (Table S2 ) and then cloned into pET28a with NdeI and XhoI sites to generate the expression plasmid pET28a-HisCpxR. The final plasmid was confirmed by sequencing. The N-terminal His-tagged CpxR protein was purified from E. coli BL21(DE3)pLysS with His-Select nickel affinity gel (Ni-NTA beads 6FF; Smart-Lifesciences) according to the manufacturer’s instructions. The purified protein presented a single band on SDS-PAGE. The CpxR protein was then desalted and concentrated using a 10-kDa Milipore ultracentrifugal filter at 4°C. The concentrated protein was divided into small portions and stored at –80°C before use. The CpxR protein concentration was measured with a BCA protein assay kit (Tiangen, China).
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