Indicated cell lines were plated in 10 cm dishes and transfected with plasmids encoding the indicated proteins. Forty-eight hours after transfection, cells were harvested and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.5% NP40 (Igepal CA630); 2 mM EDTA; 1 mM DTT; 1U benzonase; protease inhibitors; in water). Lysates were clarified by centrifugation at 15 000 g and diluted to 2 mg/ml. Lysates were then pre-cleared with 30 μl of packed Protein G Agarose Fast Flow (Millipore) for 45 min at 4°C with end-over-end rotation. For FLAG immunoprecipitation experiments, pre-cleared lysates were incubated with 25 μl of packed FLAG M2 agarose beads (Sigma-Aldrich) (or Protein G Agarose Fast Flow beads as control) and incubated for 3 h at 4°C with end-over-end rotation. For V5 immunoprecipitation experiments, pre-cleared lysates were incubated with 2 μg of V5 antibody (Cell Signaling) (or no antibody as control) overnight at 4°C with end-over-end rotation. The following day, lysates were incubated with 30 μl packed Protein G Agarose Fast Flow (Millipore) for 2 h at 4°C with end-over-end rotation. For both FLAG and V5 IP experiments, beads were washed five times with 1 ml of cold lysis buffer and eluted by boiling beads in 40 μl 1× SDS-PAGE sample buffer. Input and immunoprecipitation samples were then analyzed by western blotting.
Protein g agarose fast flow
Manufactured by Merck Group
Sourced in Germany
Protein G Agarose Fast Flow is a pre-packed chromatography resin designed for the rapid and efficient purification of antibodies and other immunoglobulins. It consists of Protein G, a bacterial protein that binds to the Fc region of immunoglobulins, covalently coupled to a highly cross-linked agarose matrix. The agarose matrix provides a robust and stable support for the immobilized Protein G, enabling high flow rates and superior binding capacity.
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Lab products found in correlation
Glycine, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Smad2 3 antibody, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (1 mentions)
Complete edta free protease inhibitor cocktail, by Merck Group (1 mentions)
Expifectamine, by Thermo Fisher Scientific (1 mentions)
Anti gapdh 14c10 rabbit mab, by Cell Signaling Technology (1 mentions)
Fugene 6 reagent, by Promega (1 mentions)
Protein g agarose fast flow beads, by Merck Group (1 mentions)
V5 antibody, by Cell Signaling Technology (1 mentions)
Flag m2 agarose beads, by Merck Group (1 mentions)
O glcnac antibody, by BioLegend (1 mentions)
11 protocols using protein g agarose fast flow
1
Immunoprecipitation of Protein Complexes
2
Purification of RG-M18 Monoclonal Antibody
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The RG-M18 mouse hybridoma cell line was established in 2001 [16 ]. To prepare RG-M18 mAB, hybridoma cells were cultured in Hybird-SFM (Gibco, USA) media in 175T flasks at 37°C with 5% CO2 supplement. Every 3 to 5 days, the supernatant was collected when the media turned yellow. Antibody was purified using Protein G Agarose Fast Flow (Merck Millipore, Germany), following the manufacturer’s instructions: supernatant was loaded onto the Protein G packaged column, and the column was then washed with a volume of ice-cold PBS equal to 10-fold that of the column. The bound antibody was eluted with 50 mM glycine (Merck, Germany) pH 2.7 (1 ml per fraction in tubes containing 10 × neutralization buffer (1 M Tris (Merck, Germany) pH 8, 1.5 M NaCl (Merck, Germany), 1 mM EDTA(Merck, Germany)). The purified antibody was stored in 50% glycerol (Merck, Germany) and 0.03% NaN3 (Merck, Germany) at -20°C.
3
Protein Analysis of Mouse Pancreas
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For protein analysis, mouse pancreas tissue was lysed and homogenized in RIPA buffer (50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 1 mmol/L ethylenediamine tetraacetic acid, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, and 1% Nonidet) containing a cocktail of protease and phosphatase inhibitors. After protein quantification, 40 μg protein was used for loading controls, and 2.5 mg protein was used for the immunoprecipitation assay by using the SMAD2/3 antibody (Cell Signaling Technology #3102; Danvers, MA) and protein G Agarose Fast Flow (EMD Millipore). Proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis and detected by Western blot analysis.
4
Immunoprecipitation of HOXA9 and ETS1
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2 × 108 of Loucy cells or total thymocytes were lysed for 30 min with 8 ml of 1× radioimmunoprecipitation assay buffer (Cell Signaling, 9806) complemented with Complete EDTA-free Protease Inhibitor Cocktail (Merck, 11873580001). The lysates were incubated overnight at 4°C with 10 µg of anti-ETS1 (C20X; Santa Cruz, sc-350) or anti-HOXA9 antibody (13C11, noncommercial production), followed by 2 h of incubation at 4°C with 20 µl of Protein G Agarose, Fast Flow (Merck, 16–266). After four washes with washing buffer (100 mM NaCl and 15 mM Tris-HCl, pH 7.8), bound proteins were eluted and detected by Western blot with anti-HOXA9 (13C11) or anti-ETS1 (C20X) antibodies.
5
Production of Anti-HIV-1 Bovine Antibody
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Antibody plasmids containing heavy chain and light chain genes were co-transfected (2:3 ratio) into Expi293F cells using Expifectamine (Thermo Fisher Scientific) according to manufacturer instructions. Supernatants containing antibodies were harvested 4 days after transfection and filtered using 0.22 μm filters. NC-Cow1 antibody was produced as an anti-HIV-1 bovine antibody control by codon optimisation of genes available from the GenBank (MF167446.1 and MF167436.1 ). Antibody supernatants were purified using Protein G Agarose Fast Flow (Merck Millipore). Antibodies were eluted from chromatography columns using 50 mM glycine (pH 2.7) and immediately neutralised by addition of 1/10 volume of 1 M Tris (pH 8.0) before being buffer exchanged into PBS, concentration using Amicon 50 kDa spin membranes (Millipore) and sterilization using 0.22 μm filters.
6
Monoclonal Antibody Production and Purification
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The monoclonal antibodies (mAbs) 17b [60 (link)], 35O22 [61 (link)], 447-52D [62 (link)], F105 [63 (link)], F240 [64 (link)], PGT121 [65 (link)], and Z13e1 [66 (link)] were obtained from the National Institutes of Health (NIH) HIV Reagent Program (HRP). Anti-Env mAbs 2F5, 2G12, 4E10, PG9, and PG16 were purchased from Polymun (Vienna, Austria). Anti-p24 mAb BC1071 and sheep polyclonal antibodies anti-p24 (D7320) and anti-gp120 (D7324) were purchased from Aalto Bio Reagents (Dublin, Ireland). Anti-GAPDH (14C10) rabbit mAb was purchased from Cell Signaling Technology (Danvers, MA, USA).
Heavy- and light-chain expression plasmids for 10E8, PGT151, and CAP256-VRC26.06 (synthesized from published protein sequences [67 (link),68 (link),69 (link)], respectively) were kindly obtained from Pantelis Poumbourios (Burnet Institute, Melbourne, Australia), and VRC01 expression plasmids were obtained from the NIH HRP [70 (link)]. Antibody heavy and light chains were coexpressed in Expi293F cells and purified using Protein G Agarose Fast Flow (MilliporeSigma) according to the manufacturer’s instructions.
Heavy- and light-chain expression plasmids for 10E8, PGT151, and CAP256-VRC26.06 (synthesized from published protein sequences [67 (link),68 (link),69 (link)], respectively) were kindly obtained from Pantelis Poumbourios (Burnet Institute, Melbourne, Australia), and VRC01 expression plasmids were obtained from the NIH HRP [70 (link)]. Antibody heavy and light chains were coexpressed in Expi293F cells and purified using Protein G Agarose Fast Flow (MilliporeSigma) according to the manufacturer’s instructions.
7
Humanized antibody expression and purification
8
Evaluating eIF4E/eIF4A Complexes by Co-IP
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eIF4E/eIF4A complexes were evaluated by co-immunoprecipitation assays under different treatments. For these immuno-precipitation assays, cellular fractions (300 µg) were incubated with the antibody anti-eIF4A (1:400) for 2 h at 4 °C. Then, immune complexes were precipitated with protein G Agarose Fast Flow (Millipore) 12 h at 4 °C, according to the previous protocol [36 (link)]. Immuno-precipitated proteins were washed 3 times, and suspended in Laemmli buffer, separated by SDS-PAGE gels and transferred to PVDF membranes for WB analysis. eIF4E detection was performed based on a previously described protocol.
9
Characterization of PDCD4/eIF4A Complex
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Characterization of the complex PDCD4/eIF4A was evaluated by co-immunoprecipitation assay under several schemes. For these immuno-precipitation assays, cellular fractions (300 µg) were incubated with the anti-eIF4A (1:400) for 2 h at 4 °C. Next, immune complexes were precipitated with Protein G Agarose Fast Flow (Millipore) for 12 h at 4 °C, according to a previous protocol [38 (link)]. Immuno-precipitated proteins were washed 3 times and suspended in Laemmli buffer, separated by SDS-PAGE gels, and transferred to PVDF membranes for WB analysis. This same process was corroborated, but now PDCD4 was immuno-precipitated, and eIF4A detection was made.
10
Immunoprecipitation of c-Jun Protein
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Nuclei separation was carried out using a buffer containing sucrose (250 mM) and imidazole (3 mM) pH 7.4, supplemented with protease and phosphatase inhibitors. Cells were scraped from culture dishes and 21 passages were performed through a 22-G syringe. For the recovery of nuclei, lysates were centrifuged at 3400 rpm for 15 min. Then, nucleus fractions (200 μg) were incubated with an anti-c-Jun antibody (1:400) for 2 h at 4 °C. Immune complexes were precipitated with protein G agarose Fast Flow (Millipore) 12 h at 4 °C. Immuno-precipitated proteins were washed 3 times and suspended in Laemmli buffer, separated by SDS-PAGE gels and transferred to PVDF membranes for Western-blot analysis. Protein detection was performed based on a previous protocol [26 (link)].
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