HEK-293T cells that were transfected with expression plasmids for 2 days were lysed in NP-40 lysis buffer (150 mM NaCl, 1 mM EDTA, 1% NP-40, 50 mM Tris-HCl (pH 8.0), supplemented with aprotinin and phenylmethylsulfonyl fluoride (PMSF). The lysate was incubated with protein G (Invitrogen) for preclearing, and further incubated overnight with anti-FLAG M2 antibody (Sigma-Aldrich). On the next day, immunoprecipitated lysate was incubated with protein G (Invitrogen), according to the manufacturer’s instructions, and, thereafter, the immunoprecipitated protein or whole cell lysate was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and polyvinylidene difluoride membrane transfer. The membrane was blocked with 5% skim milk in TBS-T (10 mM Tri-HCl (pH 7.6), 150 mM NaCl, and 0.1% Tween 20), and immunoblotted with primary antibodies against FLAG (Sigma-Aldrich) and Runx2 (Santa Cruz), followed by washing and incubation with appropriate horseradish peroxidase-linked secondary antibodies. Signals were detected with ECL solution (Millipore) and analyzed using an Azure c300 luminescent image analyzer (Azure Biosystems, Dublin, CA, USA).
Protein g
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protein G is a bacterial-derived protein that binds to the Fc region of immunoglobulins, particularly IgG. It can be used for the purification and detection of antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads protein a, by Thermo Fisher Scientific (5 mentions)
Pei max 40000, by Polysciences (4 mentions)
Dynabead, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Protein a, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
M20026, by Abmart (2 mentions)
Anti flag m2 antibody, by Merck Group (2 mentions)
Ez view anti c myc affinity gel beads, by Merck Group (2 mentions)
Mouse igg, by Merck Group (2 mentions)
Genejuice, by Merck Group (2 mentions)
Streptavidin beads, by New England Biolabs (2 mentions)
Fulvestrant from, by MedChemExpress (2 mentions)
α flag, by Merck Group (2 mentions)
α gapdh, by Santa Cruz Biotechnology (2 mentions)
Rnase a t, by Thermo Fisher Scientific (2 mentions)
αkdm2a, by Proteintech (2 mentions)
αpol 2, by Santa Cruz Biotechnology (2 mentions)
αrad21, by Abcam (2 mentions)
αlamina c, by Abcam (2 mentions)
55 protocols using protein g
1
Immunoprecipitation and Immunoblotting Analysis
2
Recombinant Hemagglutinin Protein Production
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A chimeric recombinant hemagglutinin (cHA) protein, composed of an H6 head and an H1 stalk (cH6/1) was produced as previously described (33 (link)). For use as a positive control in all the Fc assays, plasma was pooled from 5 healthy donors with high HA stalk IgG titers. From this pooled plasma IgG was isolated using Protein G (Pierce Biotechnology), according to the manufacturer’s instructions and confirmed by IgG enzyme linked immunosorbent assay (ELISA). A cross-reactive HA stalk antibody CR9114 that mediates potent Fc effector function was expressed as a positive control (34 (link)). For antibody expression, plasmids encoding heavy or light chain genes were co-transfected into HEK293F cells with PEI-MAX 40,000 (Polysciences) head-to-head. Cells were cultured for six days in 293F Freestyle media at 37°C, 10% CO2, then harvested supernatants were filtered and purified using Protein G (Thermo Scientific). Antibody concentration was quantified by nanodrop using sequence-specific extinction coefficients as determined by ProtParam (ExPASy) and confirmed by ELISA.
3
Purification of BALf IgG from IPF Patients
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BALf IgG from 6 IPF patients was purified using Protein G (Thermo Fisher Scientific, #20398). For each patient, 4 ml of BALf was diluted 1 : 1 with Protein G binding buffer (Thermo Fisher Scientific, #21011) and allowed to gravity filter through Protein G agarose. Purified IgG was eluted using 2 ml elution buffer (Thermo Fisher Scientific, #21004), and the pH was stabilised with 100 μl Tris (pH 8).
4
Glycoprotein Fractionation and Analysis
5
TAK1 Antibody Immunoprecipitation Protocol
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Total protein extract was prepared by mixing cells with lysis buffer as previously described (33 (link),34 (link)). The extract was incubated overnight in a cold room with agitation in the presence of 2 μg TAK1 antibody pre-incubated with magnetics beads coupled to protein G (Invitrogen) for 2 h. The beads were then washed seven times with 1 ml of wash buffer. The adsorbed proteins were dissociated by boiling beads for 10 min in 24 μl of Laemmli buffer and resolved by SDS-polyacrylamide gel electrophoresis. Proteins were separated by electrophoresis and electroblotted onto a nitrocellulose filter as previously described (35 (link)).
6
Neutrophil Adhesion to E-selectin and ICAM-1
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For adhesion of neutrophils to immobilized E-selectin in combine with ICAM-1 under flow, a laminar flow chamber slide (μ-Slide I0.8 Luer, ibidi, Martinsried, Germany) was filled with protein G (25 μg/mL; Invitrogen, USA) overnight. After washing with phosphate-buffered saline, the slide was filled with E-selectin (5 μg/mL; R&D system, USA) and ICAM-1 (5 μg/mL; R&D system, USA) for 2 hours and blocked with 1% bovine serum albumin (Sigma-Aldrich, USA) for 1 hour. Freshly isolated human neutrophils (5x105 cells) were preincubated without or with IND02 at RT for 1 hour, followed by perfusion at a constant flow rate of 1 dynes/cm2. The number of adherent cells (stationary over 5 seconds) was determined after 3 minutes of perfusion. The shear flow was generated by using the perfusion loop system and an air pressure pump (ibidi, Martinsried, Germany), and the images were captured by the microscope. The number of adherent cells was calculated in 10–15 random fields in a single experiment and the statistical test was calculated from 3 experiments.
7
ChIP-qPCR Analysis of TDF1-GFP Protein
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The ChIP procedure was performed as described with minor modifications. One gram of closed buds from tdf1 gTDF1pro:TDF1-GFP complemented plants frozen in liquid nitrogen was crosslinked in 0.4 M sucrose-1% (v/v) formaldehyde buffer. Nuclei were isolated with extraction buffer and lysed with lysis buffer. The chromatin was sheared with sonication, resulting in most DNA fragments having a size between 200 and 800 bp. After pre-absorption using pre-immune serum with sheared salmon sperm DNA/protein A agarose mix (Millipore, USA) for 1 h, the DNA–protein complex was immunoprecipitated at 4 °C overnight using an anti-GFP monoclonal antibody (Millipore, USA) (1:100 dilution). Seventy microliters of magnetic beads coupled with protein G (Invitrogen, USA) were added to precipitate the antibody–protein/DNA complexes. After washing, the samples were incubated at 65 °C overnight to reverse the crosslinking. The co-precipitated DNA was purified and analyzed by qPCR as described above. Under the same conditions, the ΔCt values (Ct of each sample—Ct of the No antibody control) were calculated and reported 2−ΔCt as the fold enrichment. Primer sequences are listed in Supplemental Data Set S1 .
8
Genome-wide Profiling of Chromatin Modifications
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ChIP-seq is a method used to analyze protein interactions with DNA. ChIP-seq combines with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. 106 differentiated cells from Day 26, Day 39, and Day 55 stages were cross-linked using 1% formaldehyde. The lysate with sodium dodecyl sulfate-based reagents and chromatin was sonicated for 18 cycles (60 s On, 60 s Off) using Bioruptor. The sonicated samples were immunoprecipitated using magnetic beads 25 μL protein A (Invitrogen cat.10002D) and 25 μL protein G (Invitrogen cat.10004D). The samples were reverse crosslinked using Proteinase K overnight at 650°C. The sonicated fragments were 300-500bp in size. The DNA fragments were purified using the phenol-chloroform protocol. ChIP was performed using antibodies against H3K27ac (Abcam Ab4729) and H3K27me3 (Abcam Ab4729). Prepared libraries from ChIP and input DNA were sequenced using the HiSeq instrument (Illumina, United States). The ChIP libraries were analyzed and mapped to hg19 using BWA (Li and Durbin, 2009 (link)) and peaks were called using MACS (Zhang et al., 2008 (link)).
9
Protein G-Coated Plate for OPN-Fc Binding
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The 24-well microtiter plate was pre-coated on the surface using 50 μg/ml Protein G (Invitrogen, Thermo Fisher Scientific, USA) 250 μl per well and incubated overnight at room temperature. The plate was blocked with 10 mg/ml BSA for 2 h at room temperature before washing with PBS 3 times. The plate was coated with 50, 100, 200 and 300 ng of plant-produced OPN-Fc fusion protein and incubated 2 h at room temperature. The hPDL cells were seeded in 24- well microtiter plate at a density of 8 × 104 cells per well. After 24 h treatment, RNA was extracted for real-time polymerase chain reaction (RT-PCR) analysis.
10
Investigating Htt-Hsp90 Interactions in SH-SY5Y Cells
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To determine the interaction between nHtt or mHtt with Hsp90, lysates from differentiated SH-SY5Y cells were transiently transfected with expression vectors of green fluorescent proteins (GFPs), GFP-480-68Q or GFP-480-17Q (control) for 15 h. Cells were prepared in lysis buffer and subjected to immunoprecipitation (IP) with anti-Hsp90α antibody (Abcam, AB133491) or anti-HA antibody (Sigma, H9658) as a control; then, the membrane was blotted with Htt antibody. To determine whether Hsp90α is associated with REST, differentiated SH-SY5Y cells were prepared in a lysis buffer containing HEPES (20 mM, pH 7.6), NaCl (150 mM), glycerol (2.5%), Nonidet P-40 (1%), EDTA (13.4 mM), EGTA (2 mM), NaF (50 mM), Na3VO4 (1mM), phenylmethylsulfonyl fluoride (1 mM), and a protease inhibitor cocktail. Lysates were subjected to immunoprecipitation with 2.5 μg of anti-REST, anti-Hsp90α, or nonimmune anti-HA antibody for 20 h at 4°C. Cells were then added with protein G (Invitrogen, 15920–010), incubated for 4 h, washed twice with lysis buffer without Nonidet P-40, and eluted with SDS-PAGE sample loading buffer. Immunoprecipitates were subjected to SDS-PAGE analysis followed by Western blot analysis with anti-Htt, anti-REST, or anti-Hsp90.
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