Protein G-sepharose, by Pierce - Product detail - Pubcompare (2024)

Manufactured by Pierce

Sourced in France

Protein G-Sepharose is a chromatography resin composed of recombinant Protein G coupled to Sepharose beads. Protein G is a bacterial cell wall protein that binds to the Fc region of immunoglobulins, enabling the purification of antibodies from complex biological samples.

Automatically generated - may contain errors

Lab products found in correlation

PR-619, LifeSensors (2 mentions) Hm79b, BD (2 mentions) Mini-PROTEAN TGX gel, Bio-Rad (2 mentions) Anti-DDX21, Novus (2 mentions) Anti-FLAG, Sigma-Aldrich (2 mentions) Vibra-Cell sonicator, Sonics (1 mentions) N-ethylmaleimide (NEM), Sigma-Aldrich (1 mentions) Protein A-Sepharose, Pierce (1 mentions) P4D1, Santa Cruz Biotechnology (1 mentions) F(ab)2 goat anti–mouse Ig (H+L), Jackson ImmunoResearch (1 mentions) Protease inhibitor cocktail tablets, Roche (1 mentions) Immun-Blot PVDF membrane, Bio-Rad (1 mentions) PVDF membranes, Millipore (1 mentions) Anti-NOP58, Bethyl Laboratories (1 mentions) Anti-fibrillarin, Cell Signaling (1 mentions) Anti-HEXIM1, Bethyl Laboratories (1 mentions) Anti-cyclin T1, Santa Cruz Biotechnology (1 mentions) Anti-CDK9, Santa Cruz Biotechnology (1 mentions) Immun-Blot, Bio-Rad (1 mentions) Anti-ubiquitin (P4D1), Santa Cruz Biotechnology (1 mentions)

9 protocols using Protein G-sepharose

Testis tissue was homogenized in 750 μl lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Triton-X, 1 mM Na3VO4, 5 mM NaF, 1 mM PMSF, 1×Protease inhibitor cocktail). Protein extracts were sheared on ice with 15 1-s pulses at 30% amplitude using a Vibra-Cell sonicator (Sonics) and incubated for 30 min on a rotating wheel at 4°C. After centrifugation at 16,000 

g

for 15 min, the supernatant was incubated with 5 μl of anti-ubiquitin ON at 4°C. Immune complexes were precipitated with protein G-sepharose (Pierce, Thermo Fisher Scientific) for 4 h at 4°C and the bead pellets were washed five times with ice-cold lysis buffer. 1×Lämmli was added to the pellets and heated for 5 min at 92°C. After centrifugation the supernatant was subjected to SDS-page.

Milenkovic A., Schmied D., Tanimoto N., Seeliger M.W., Sparrow J.R, & Weber B.H. (2019). The Y227N mutation affects bestrophin-1 protein stability and impairs sperm function in a mouse model of Best vitelliform macular dystrophy. Biology Open, 8(7), bio041335.

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The hybridoma (ref. ATCC PTA-9008) derived CD44-specific monoclonal antibody was purchased from ATCC (France) and the mAbs were purified on protein G-sepharose (Pierce Chemical). Reagents used for in vivo treatment were diluted in PBS and sterilized using a sterile 0.22 μm pore-size filter.

Molejon M.I., Tellechea J.I., Loncle C., Gayet O., Gilabert M., Duconseil P., Lopez-Millan M.B., Moutardier V., Gasmi M., Garcia S., Turrini O., Ouaissi M., Poizat F., Dusetti N, & Iovanna J. (2015). Deciphering the cellular source of tumor relapse identifies CD44 as a major therapeutic target in pancreatic adenocarcinoma. Oncotarget, 6(10), 7408-7423.

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For serum protein competitive binding analysis, first, 1×1010 particles of AAV8/luc vector were incubated with HSA at different dilutions (1:100, 1:1000, 1:10000, 1:10000 of physiological concentration) or PBS on ice for 2hr, and then LDL, or TRF or ApoB at d̵ 100-fold dilution of physiological concentration was added to the AAV-HSA mixture and incubated on ice for 2hr Next, the antibodies of TRF or ApoB conjugated to Protein G Sepharose (Pierce and followed their manual) were added and incubated at 4 °C overnight. Then, TRF or LDL or ApoB specific binding serum protein-AAV complex was pulled down. The complex was washed three times with cold PBS and transferred to a new micro-tube. DNA from the complex was extracted and applied for qPCR to determine the AAV genome copy number per cell using luc specific primers. To study the effect of serum proteins’ receptors on transduction of AAV8 or AAV8 serum protein complex, 5×108 particles of AAV8/luc were incubated with serum proteins (4μg transferrin or 2μg LDL or PBS) individually at 4 C° for 1hr, then 4μg transferrin-receptor protein or LDL-receptor protein were added for another hour at 4 °C. The mixture was applied to 5×104 Huh7 cells, which were seeded on a 48-well plate in 300 ul of serum-free medium, X-vivo 10. 48hr later, luciferase activity from cell lysate was measured with a Wallac1420 Victor 2 automatic plate reader.

Pei X., He T., Hall N.E., Gerber D., Samulski R.J, & Li C. (2018). AAV8 Virions Hijack Serum Proteins to Increase Hepatocyte Binding for Transduction Enhancement. Virology, 518, 95-102.

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Splenic B cells were purified by negative selection as described above. Cells were stimulated with 20 µg/ml F(ab)2 goat anti–mouse Ig (H+L) (Jackson ImmunoResearch Laboratories) at 37°C for indicated times. Cell aliquots were lysed in 1% NP40 buffer (150 mM NaCl, 10 mM Tris HCl pH 7.7, 5 mM EDTA, 0.4 mM sodium orthovanadate, and 10 mM sodium pyrophosphate) containing mini EDTA-free protease inhibitor cocktail tablets (Roche), phenylmethylsulfonyl fluoride (PMSF)(Sigma-Aldrich), 20 mM N-Ethylmaleimide (NEM) (Sigma-Aldrich), 10 mM 1,10-phenatholine monohydrate (OPD) (Sigma-Aldrich), and 50 µM PR-619 (LifeSensors). Lysates were clarified by centrifugation at 4°C. Lysates were precleared with protein A–Sepharose (Pierce), incubated with primary antibodies specific for Igβ (Hm79b, BD Biosciences) and captured with protein G–Sepharose (Pierce). Lysates or immunoprecipitates were resolved on a 4–15% Mini-Protean TGX gel (Bio-Rad) and transferred onto Immun-Blot PVDF membrane (Bio-Rad). Membranes were probed with antibodies specific for ubiquitin (P4D1, Santa Cruz), Igβ [24] (link) or Cbl-b.

Veselits M., Tanaka A., Lipkowitz S., O'Neill S., Sciammas R., Finnegan A., Zhang J, & Clark M.R. (2014). Recruitment of Cbl-b to B Cell Antigen Receptor Couples Antigen Recognition to Toll-Like Receptor 9 Activation in Late Endosomes. PLoS ONE, 9(3), e89792.

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Myeloma cells were appropriately treated with GBT (50nM) and lysed with RIPA buffer. Clarified lysates were precleared with Protein G-Sepharose (Pierce, Rockford, IL), and then immuno-precipitated with anti-HA, anti-flag, or antic-Myc absorbed to Protein G-Sepharose. Proteins were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, MA) and blotted with anti-flag, anti-HA, or anti-Ubiquitin antibodies, respectively. 293T cells were transfected with different plasmids encoding FLAG-WWP2, His-c-Myc, or HA-ubiquitin.

Yu Z., Li T., Wang C., Deng S., Zhang B., Huo X., Zhang B., Wang X., Zhong Y, & Ma X. (2016). Gamabufotalin triggers c-Myc degradation via induction of WWP2 in multiple myeloma cells. Oncotarget, 7(13), 15725-15737.

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HEK293 nuclear extracts were prepared as described previously35 (link). For immunoprecipitations, extracts were incubated overnight with 3 μg of the desired antibody pre-bound to protein G-sepharose (Pierce). In some case protein extracts were treated with RNaseA (20 μg ml−1). Immunocomplexes were eluted in 2× Laemmli buffer and resolved in an 8% acrylamide gel. For western blots the following antibodies were used according to manufacturer instructions: anti-NOP58 (Bethyl A302-718A); anti-fibrillarin (Cell Signaling C13C3); anti-DKC1 (Gene Tex GTX109000); anti-Flag (Sigma); anti-DDX21 (Novus Biologicals NB100-1781); anti-LARP7 (a gift from D. H. Price); anti-CDK9 (Santa Cruz Biotechnology sc-484); anti-cyclinT1 (Santa Cruz Biotechnology sc-10750); and anti-HEXIM1 (Bethyl A303-113A). All antibodies have been previously validated unless otherwise specified.

Calo E., Flynn R.A., Martin L., Spitale R.C., Chang H.Y, & Wysocka J. (2014). RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature, 518(7538), 249-253.

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Splenic B cells were purified by negative selection as described above. Cells were stimulated with 20 µg/ml F(ab)2 goat anti–mouse IgG and IgM (H+L; Jackson ImmunoResearch Laboratories) at 37°C for the indicated times. For Western blotting, cell aliquots were lysed in modified radioimmunoprecipitation assay buffer containing protease inhibitors and PMSF as described previously (Kabak et al., 2002 (link)). For ubiquitin immunoblots, cells were lysed as above with the addition of 20 mM N-ethylmaleimide (Sigma-Aldrich), 10 mM 1,10-phenatholine monohydrate (Sigma-Aldrich), and 50 µM PR-619 (LifeSensors). Cellular lysates were precleared with protein G–Sepharose (Pierce), incubated with primary antibodies specific for Igβ (Hm79b; BD Biosciences), and captured with protein G–Sepharose (Pierce). Lysates or immunoprecipitates were resolved on a 4–15% Mini-Protean TGX gel (Bio-Rad) and transferred onto polyvinylidene fluoride membrane (Immun-Blot; Bio-Rad). Antibodies with the indicated specificities were used: ERK1/2 (137F5; Cell Signaling), p38 (9212; Cell Signaling), CD19 (3574; Cell Signaling), actin (C4; Millipore), phosphotyrosine (4G10; Millipore), CD79b (Luisiri et al., 1996 (link)), antiubiquitin (P4D1; Santa Cruz), and phospho-T202/Y204 ERK1/2 (197G2), pT180/T182 p38 (9244S), pT458 p85 (4228), pS473 Akt (193H12), and Y531 pCD19 (3571; all from Cell Signaling).

Veselits M., Tanaka A., Chen Y., Hamel K., Mandal M., Kandasamy M., Manicassamy B., O’Neill S.K., Wilson P., Sciammas R, & Clark M.R. (2017). Igβ ubiquitination activates PI3K signals required for endosomal sorting. The Journal of Experimental Medicine, 214(12), 3775-3790.

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1 μg of HEL46-61–I-Ak-btn MHC class II monomer (N.I.H. Tetramer Core Facility) in 100 μl of buffer was immunoprecipitated overnight at 4°C with 5 μg of the indicated mAb plus 100 μl of 10% Protein G-Sepharose (Pierce, Catalog #: 20398). IPs were washed and analyzed by SDS-PAGE and western blot using streptavidin –HRP (Pierce Catalog #: 21124) at 1:25,000 in borate buffer 0.1% BSA. Blots were developed with SuperSignal West Dura ECL substrate. The Aw3.18 (Dadaglio et al., 1997 (link)) and C4H3 (Zhong et al., 1997 (link)) mAbs are specific for HEL46-61–I-Ak complexes and were used to demonstrate the presence of properly assembled peptide-class II monomers.

Drake L.A, & Drake J.R. (2016). A Triad of Molecular Regions Contribute to the Formation of Two Distinct MHC Class II Conformers. Molecular immunology, 74, 59-70.

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Nuclear extracts were prepared according to the Dignam and Roeder protocol32 . For immunoprecipitations, extracts were incubated overnight with 3–5 μg of the desired antibody pre-bound to Protein G Sepharose (Pierce). In some cases, protein extracts were treated with RNaseA (20 μg ml−1). Immunocomplexes were eluted in 2× Laemmli buffer and resolved in a 4–20% pre-casted tris-glycine gel (Life Technologies). For western blots, the following antibodies were used according to the manufacturer’s instructions: anti-GFP (Thermo Fisher Scientific A-6455), anti-TCOF1 (Novus Biologicals NBP1-86909 and Abnova H00006949-B01P), anti-Flag (Sigma-Aldrich), anti-DDX21 (Novus Biologicals NB100-1781), anti-p53 (Vector Laboratories VP-P953), anti-ACTIN (Abcam ab49900). All antibodies had been previously validated unless otherwise specified.

Calo E., Gu B., Bowen M.E., Aryan F., Zalc A., Liang J., Flynn R.A., Swigut T., Chang H.Y., Attardi L.D, & Wysocka J. (2018). Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders. Nature, 554(7690), 112-117.

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Protein G-sepharose, by Pierce - Product detail - Pubcompare (2024)
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