The total IgG was purified from the FF by using Protein G-agarose (Sigma Aldrich, USA) according to the manufacturer instructions. Briefly, 100 µl of the FF was diluted 10x in 20 Mm NaH2PO4 (pH 7.0; Sigma Aldrich, USA) and incubated with 50 µl of homogenous Protein G-agarose suspension overnight at 4 °C. After incubation, the remaining FF supernatant, which contained FF total proteomes separated from the main IgG fraction, was stored at -80°C, while the agarose beads with bound IgG were first washed with 3x 200 µl of 20 Mm NaH2PO4 followed by 5x 200 µl of 20 mM NaH2PO4/ 150 Mm NaCl (pH 7.0; SigmaAldrich, USA) to remove non-specific binding. The bound IgG was released from the Protein G-agarose by incubation in 100 µl of 100 mM HCl (SigmaAldrich, USA) for 5 min on RT. The supernatant containing released FF IgG was neutralised by addition of 20 µl of 500 mM NaOH (SigmaAldrich, USA). The isolated FF IgG and FF total protein concentrations were determined by using Qubit TM quantitation platform (Invitrogen, USA). To assess the purity of isolated FF IgG and measured concentrations of isolated IgG and FF total proteomes, the amounts of each sample corresponding to 3 µg of FF IgG and to 50 µg of FF total proteomes were subjected to 10% SDS-PAGE at 110V for 1.5 h (Supplementary Figure ).
Protein g agarose
Manufactured by Merck Group
Sourced in United States, Germany
Protein G agarose is a laboratory reagent used for the purification and isolation of antibodies and antibody-containing samples. It consists of the Protein G ligand, which binds specifically to the Fc region of immunoglobulins, immobilized on an agarose bead support. Protein G agarose facilitates the capture and separation of antibodies from complex biological samples.
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Lab products found in correlation
Anti flag m2 affinity gel, by Merck Group (6 mentions)
Protein a and protein g magnetic beads, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Protein g agarose, by Santa Cruz Biotechnology (4 mentions)
Nigericin, by Merck Group (3 mentions)
Anti human caspase 1, by Cell Signaling Technology (3 mentions)
Mitosox, by Thermo Fisher Scientific (3 mentions)
Mitotracker, by Thermo Fisher Scientific (3 mentions)
Adenosine triphosphate (atp), by Merck Group (3 mentions)
Ab1220, by Abcam (3 mentions)
Anti flag, by Merck Group (3 mentions)
Protein a agarose, by Roche (3 mentions)
Mg132, by Avantor (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Sappα, by Fortrea (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Flag peptide, by Merck Group (2 mentions)
Methionine free dmem, by Thermo Fisher Scientific (2 mentions)
Click it hpg, by Thermo Fisher Scientific (2 mentions)
140 protocols using protein g agarose
1
Protein G-based IgG Purification Protocol
2
EGF-Induced Protein Interaction Analysis
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Cells were treated with EGF (100 ng/mL) or vehicle for 10 min. Then cells were lysed in co-IP buffer (10 mM HEPES pH 8.0, 100 mM NaCl, 0.1 mM EDTA, 20% glycerol, 0.2% NP-40, Halt™ Phosphatase Inhibitor Cocktail, and Halt Protease Inhibitor Cocktail (Thermo Fisher)). Lysates were centrifuged and cleared by incubation with 25 μl of Protein G agarose (Millipore) for 1.5 h at 4 °C. The pre-cleared supernatant was subjected to IP using the indicated primary antibodies at 4 °C overnight. Then, the protein complexes were collected by incubation with 30 μl of Protein G agarose (Millipore) for 2 h at 4 °C. The collected protein complexes were washed 6× with co-IP buffer and analyzed by western blotting.
3
Immunoprecipitation and Immunoblot Analysis
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After preclearing for 30 minutes with protein G agarose (Millipore), antibodies specific for Gαq/11, Gβ, or PI3K (1:100; Santa Cruz Biotechnology) or IgG (2 μg, Sigma-Aldrich Co, St. Louis, MO, USA) were added before overnight incubation at 4°C, followed by precipitation for 2 hours with protein G agarose. The beads were washed three times with RIPA lysis buffer, boiled in sample buffer, and the protein were resolved by 8% SDS-PAGE before performing immunoblot analysis of the indicated proteins.
4
Protein-Protein Interaction Assay
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Tagged Psmd2 full-length protein and the RID domain of Rpgrip1l were transiently overexpressed in NIH3T3 cells. After 24 h of expression, cells were lysed on ice in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and 0.25% Na-deoxycholate) supplemented with complete protease inhibitor cocktail (Roche) and PMSF (Sigma-Aldrich). Lysates were precleared with protein G–agarose (EMD Millipore) overnight followed by an overnight incubation with anti-FLAG or anti-Myc protein G–agarose. Afterward, beads with bound protein complexes were washed in lysis buffer, taken up in 4× sample buffer (50 mM Tris-HCl, pH 6.8, glycerol, 2% SDS, 20% β-mercaptoethanol, and bromophenol blue), and heated for 10 min at 95°C. Beads were precipitated by centrifugation. Supernatant was run on a SDS-PAGE gel and then immunoblotted. Coimmunoprecipitation experiments were repeated three times.
5
TRAF6 Immunoprecipitation from RAW264.7 Cells
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RAW264.7 cells were starved in serum-free medium for 6 h and then treated with the indicated prescriptions. The cells were then washed with cold PBS and lysed in RIPA buffer with protease inhibitor cocktail (cOmplete; Roche, Basel, Switzerland). Cell lysates were precleared with 30 µL of protein G-agarose (EMD Millipore, Bedford, MA, USA), and the precleared lysates were incubated with the TRAF6 antibody at 4 °C for 18 h. Next, protein G-agarose beads were added to each lysate and incubated 4 °C for 2 h. Subsequently, the beads were washed three times with lysis buffer, and the bound proteins were separated using SDS-PAGE followed by Western blotting with the specific antibodies.
6
CXCR2 Ubiquitination Assay in HEK-293T Cells
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HEK-293T cells were transfected with mock, CXCR2-WT or K327R (R1) cDNA plasmids. 24 hours post-transfection, cells were treated as indicated and were further lysed (25 mM Hepes pH7.4, 130 mM NaCl, 10% glycerol, 8 mM β − glycerophosphate, 0.2% Igepal, 1 mM DTT, 1 mM NaVO4, 1 mM NaF, protease inhibitors, 1% SDS and 1 μl benzonase (250 U/μl, Novagen)) on ice. After 30 minutes, SDS was diluted to reach a final concentration of 0.1% and samples were precleared with Protein G agarose (Sigma) for 30 min and then incubated with antibody to CXCR2 and Protein G agarose for 1 hour at 4°C. Ubiquitination status were analyzed by immunoblot using antibody against ubiquitin.
7
VDAC Binding Assay Protocol
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To assess VDAC binding, cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, 5 mM NaF, 1 mM Na3VO4) supplemented with protease inhibitor cocktail (Sigma). Cell lysates (1 mL) were precleared by incubation with 100 μL of protein G agarose (Sigma) at 4°C for 10 min and blocked at RT for 1 h with 10 μM VDAC-binding membrane domain peptide (Calbiochem). The resultant lysate (500 μg) was immune precipitated at 4°C for 2 h with 2 μg rabbit polyclonal anti-VDAC1/2/3 (Santa Cruz), and immune complexes were captured by overnight incubation with 100 μL of protein G agarose at 4°C. Purified VDAC-protein G agarose bead complexes were washed with cold PBS and incubated at RT for 50 min with recombinant HK protein (Sigma) at indicated pH. Beads were washed with PBS and bound proteins used for western blots for HK-1, HK-2, and VDAC.
8
BCL-2 Protein Immunoprecipitation
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Cells were washed once with PBS and lysed in 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS) buffer containing protease and phosphatase inhibitors and PMSF for 30 min on ice. Debris was removed by centrifugation at 14,000 rpm at 4 °C for 15 min. Total protein concentration was determined using Bio-Rad Protein Assay Dye Reagent Concentrate (#5000006 ). 340 µL lysates containing 113 µg protein were precleared by incubating for 1 h at 4 °C with 50 µL of Protein G Agarose (Millipore#16-266), prewashed with PBS and CHAPS buffer. Precleared lysates were collected by centrifugation at 5000 rpm for 2 min at 4 °C and incubated with rotation overnight at 4 °C with 50 µL of Protein G Agarose, preincubated with 7 µL of BCL-2 (CST #4223S) antibody overnight. Pelleted matrix was washed twice with 500 μL ice-cold PBS, and proteins were eluted after boiling at 95 °C for 5 min in 30 µL of 2× electrophoresis sample buffer containing β-mercaptoethanol.
9
Immunoprecipitation of NPCs using Antibody
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Immunoprecipitation using NPCs was performed by following report [22 (link)]. NPCs were lysed with LB buffer for 15 min and the lysates were centrifuged at 1500 g for 5 min. The supernatant was incubated with antibody for 24 hour at 4℃. After incubation, the sample was incubated with Protein G Agarose (Millipore) for 24 hour at 4℃. After incubation, the supernatant was discarded after centrifugation. The agarose beads in the pellet were washed using LB buffer. The pellet was used for Western blot experiments. For negative control experiments, all procedures were identically handled without any antibody.
10
Quantifying APP Secretase Activity
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Cells were incubated with justicidin A for 8 h, and the media were concentrated using Amicon Ultra 30K centrifugal filters (Millipore). The concentrated media was immunoprecipitated with an APP antibody recognizing the N-terminus (abcam, MA, USA) and Protein G Agarose (Millipore). The immunoprecipitated samples were washed with PBS, and probed for sAPPα (Covance, NJ, USA) and sAPPβ (Covance) using Western blot.
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