Protein a or protein g sepharose beads

Manufactured by GE Healthcare

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Protein A or protein G sepharose beads are laboratory equipment used for the purification and isolation of antibodies and other proteins. They are composed of protein A or protein G immobilized on a sepharose support matrix. Protein A and protein G are bacterial cell wall proteins that have a high affinity for the Fc region of immunoglobulin molecules, allowing them to effectively capture and separate antibodies from complex samples.

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11 protocols using protein a or protein g sepharose beads

Immunoblot and Immunoprecipitation Assays Protocol

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Immunoblot and immunoprecipitation assays were performed as described previously (29 (link)). Briefly, NETN buffer [20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Nonidet P-40, 100 mM NaCl], 10 mM NaF, 50 mM β-glycerophosphate, and 1 mg/ ml each of pepstatin A and aprotinin were used to lyze the cells. Cell lysates were centrifuged at 10,000 rpm for 15 min and the supernatant was removed and incubated with 2 μg of the indicated antibody and 20 μl protein A or protein G Sepharose beads (GE Healthcare, MA) overnight at 4 °C. Immunoprecipitates were centrifuged at 8000 rpm for 1 min, washed twice with cold NETN buffer and boiled with 1× Laemmli buffer for 10 min. The samples were then separated by SDS–PAGE and transferred to PVDF membranes using the semi-dry method (Trans-Blot^® Turbo^™ Transfer System, Bio-Rad, CA). PVDF membranes were incubated with 5% milk for 1 h, followed by incubation with the indicated primary antibody overnight at 4^oC. On the following day, membranes were washed with PBST buffer x3, and then incubated with goat anti-rabbit HRP (Jackson Immunoresearch, AB_2313567, RRID: AB_2313567) or goat anti-mouse HRP (Jackson Immunoresearch, PA; AB_10015289, RRID: AB_10015289) secondary antibodies for 1 hr. After washing x3 in PBST buffer, the membranes were incubated with ECL and the signal was detected using an Azure imaging system (Dublin, CA) or by X-ray film.

Bouvier M, & Wiley D.C. (1996). Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.. Proceedings of the National Academy of Sciences of the United States of America, 93(10), 4583-4588.

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Immunoprecipitation and Western Blot

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Cells harvested in the above mentioned lysis buffer were incubated with the primary antibody overnight at 4 °C which was followed by an incubation of 90 min with protein A or protein G sepharose beads (GE Healthcare, Uppsala, Sweden). Beads were then washed three times with chilled PBS. Immunoprecipitates were eluted through boiling the beads in SDS-PAGE sample buffer for 10 min, subjected to SDS-PAGE followed by western blot analysis.

Nailwal H., Sharma S., Mayank A.K, & Lal S.K. (2015). The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43. Cell Death & Disease, 6(5), e1768-.

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Affinity Purification of Proteins

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Anti-HA affinity purification has been described previously (Tirard et al., 2012 (link); Tirard and Brose, 2016 (link)). For immunoprecipitation of specific proteins (syntaxin1a, gephyrin, GluK2, synapsin1, RIM1) from whole brain or from HEK cells, tissue or cells were homogenized in lysis buffer (150 mM NaCl, 1% Triton X-100, 10 mM Tris, pH 7.6) containing protease inhibitors (1 µg/ml aprotinin, 0.5 µg/ml leupeptine, 17.4 µg/ml PMSF) and 20 mM NEM, sonicated, and ultracentrifuged at 100,000 x g for 1 hr at 4°C. The resulting supernatant was preincubated with either Protein A or Protein G Sepharose beads (GE Healthcare, Feibrug, Germany) and antibodies (specific IgG or control IgG from the same species) or anti-HA beads (Sigma-Aldrich, Taufkirchen, germany) were added to the supernatant. After incubation for 4 hr at 4°C on a rotating wheel, the beads were pelleted and washed repeatedly in lysis buffer. Bound material was eluted directly into SDS-PAGE sample buffer.

Daniel J.A., Cooper B.H., Palvimo J.J., Zhang F.P., Brose N, & Tirard M. (2017). Analysis of SUMO1-conjugation at synapses. eLife, 6, e26338.

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Co-immunoprecipitation and Ubiquitination Assays

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For coimmunoprecipitation experiments cells were lysed in ELB (0.25 M NaCL, 0.5% NP-40, 50 mM HEPES [pH 7.3]) supplemented with proteasome inhibitors (Complete; Roche). Cell lystates (500 μg to 1 mg) were incubated overnight with 1 μg of the indicated antibodies conjugated. Subsequently the lysates were then incubated for up to 6 h with protein A or protein G sepharose beads (GE Healthcare), washed three times in ELB buffer and separated out on SDS-PAGE gels. For in vivo ubiquitination experiments TβRI (5 μg) or TβRII (5 μg) was co-transfected with HA-Ubiquitin (5 μg) and FLAG-OTUD4 (5 μg), FLAG-OTUD4 DD (5 μg), or a control vector. For loss-of-function experiments FLAG-TβRI C.A. (2 μg) were co-transfected with HA-Ubiquitin (5 μg) and pRS OTUD4 B, pRS OTUD4 C (10 μg) or control vector. For the FLAG-TβRI C.A. experiment, after 72 h MG132 (5 μM) was added, incubated overnight, and cells were lysed in ELB buffer.

Jaynes P.W., Iyengar P.V., Lui S.K., Tan T.Z., Vasilevski N., Wright S.C., Verdile G., Jeyasekharan A.D, & Eichhorn P.J. (2020). OTUD4 enhances TGFβ signalling through regulation of the TGFβ receptor complex. Scientific Reports, 10, 15725.

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Immunoprecipitation of CFTR Protein

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Antibodies against CFTR were pre-incubated with protein-A or protein-G Sepharose beads (GE Healthcare) for 15 min at 4 °C before adding protease-treated or non-treated lysates. The lysates were added to the antibody-beads mixtures and incubated at 4 °C for either 3 h or overnight. The beads were washed twice for 15 min at room temperature. A list of immunoprecipitation conditions is provided in the supplementary materials (Table S8). Beads were resuspended in 10 μl 10 mM Tris–HCl pH 6.8 containing 1 mM EDTA, and immune complexes were eluted by adding 10 μl 2 × reducing Laemmli sample buffer (final concentration: 200 mM Tris–HCl pH 6.8, 3% SDS, 10% glycerol, 1 mM EDTA, 0.004% bromophenol blue, and 25 mM DTT) and heating for 5 min at 55 °C.

Im J., Hillenaar T., Yeoh H.Y., Sahasrabudhe P., Mijnders M., van Willigen M., Hagos A., de Mattos E., van der Sluijs P, & Braakman I. (2023). ABC-transporter CFTR folds with high fidelity through a modular, stepwise pathway. Cellular and Molecular Life Sciences, 80(1), 33.

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Coimmunoprecipitation and Ubiquitination Assays

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For coimmunoprecipitation experiments, cells were lysed in ELB (0.25 M NaCL, 0.1% NP-40, and 50mM Hepes, pH 7.3) supplemented with proteasome inhibitors. Cell lystates (500 µg–1 mg) were incubated for 2 h to overnight with 2 µg of the indicated antibodies conjugated to protein A or protein G sepharose beads (GE Healthcare), washed three times in ELB buffer, and separated out on SDS-PAGE gels. In vivo deubiquitination experiments were performed as in Kit et al. (Kit Leng Lui et al., 2017 (link)). In brief, BRAF (5 µg) alone or along with USP28 shRNA (20 µg) was cotransfected with HA-Ubiquitin (5 µg) or a control vector. For endogenous ubiquitination experiments, BRAF (5 µg) was cotransfected with Myc-tagged FBW7 (5 µg) or Myc-tagged FBW7 mutant (5 µg) or FBW7 shRNA (20 µg) or control vectors. After 72 h MG132 (5 µM) was added, incubated overnight, and cells were lysed in ELB buffer. The level of ubiquitination was measured using Ubiquitin antibody (Santa Cruz) or HA antibody.

Saei A., Palafox M., Benoukraf T., Kumari N., Jaynes P.W., Iyengar P.V., Muñoz-Couselo E., Nuciforo P., Cortés J., Nötzel C., Kumarakulasinghe N.B., Richard J.L., Bin Adam Isa Z.F., Pang B., Guzman M., Siqin Z., Yang H., Tam W.L., Serra V, & Eichhorn P.J. (2018). Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies. The Journal of Experimental Medicine, 215(7), 1913-1928.

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Immunoprecipitation of Ubiquitinated SMURF2

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For co-immunoprecipitation experiments, cells were lysed in ELB (250 mM NaCl, 0.5% NP-40, 50 mM HEPES, pH 7.3) supplemented with protease inhibitors. Cell lysates were incubated for overnight with the indicated antibodies, then conjugated to protein A or protein G sepharose beads (GE Healthcare), washed three times in ELB buffer, boiled in sample buffer and separated out on SDS–PAGE gels. When appropriate cell lysates were immunoprecipitated with ANTI-FLAG M2 affinity resin (Sigma). Wild-type Myc-tagged SMURF2 (5 μg) or corresponding relevant mutants were co-transfected with HA-ubiquitin (3 μg). After 72 h MG132 (2 μM) was added, incubated overnight and cells were lysed in ELB buffer. SMURF2 was immunoprecipitated with Myc antibodies and bound proteins were resolved by SDS–PAGE gel for further processing by immunoblotting^{73 (link)}.

Sim W.J., Iyengar P.V., Lama D., Lui S.K., Ng H.C., Haviv-Shapira L., Domany E., Kappei D., Tan T.Z., Saei A., Jaynes P.W., Verma C.S., Kumar A.P., Rouanne M., Ha H.K., Radulescu C., ten Dijke P., Eichhorn P.J, & Thiery J.P. (2019). c-Met activation leads to the establishment of a TGFβ-receptor regulatory network in bladder cancer progression. Nature Communications, 10, 4349.

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Immunoprecipitation Protocol with Detailed Lysis Conditions

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For immunoprecipitations, cells were lysed in lysis buffer (25 mM TrisHCl, pH 7.4, 1% (v/v) Nonidet P-40 (NP-40, AppliChem, Darmstadt, Germany), 150 mM NaCl, protease inhibitors (Complete Protease Inhibitor Cocktail; Roche, Indianapolis, IN), 5% glycerol ()AppliChem #A2926) and phosphatase inhibitors (PhosSTOP TM , Roche, Indianapolis, IN), 2 mM sodium orthovanadate) for 20 min with overhead rotation at 4°C followed by centrifugation (15.000 rpm, 20 min at 4°C). Postnuclear supernatants were incubated with 3 μg of antibodies coupled to protein A-or protein G-Sepharose beads (GE Healthcare, Solingen, Germany) for 4 h at 4°C. Beads were washed five times with lysis buffer, bound proteins were eluted by boiling in 3x SDS-sample buffer/150 mM DTT. Eluted proteins were separated by SDS-PAGE and analyzed by Western blotting with near-infrared fluorescence detection (Odyssey Infrared Imaging System Application Software Version 3.0 and IRDye 800CW-conjugated antibodies; LI-COR Biosciences, Bad Homburg, Germany).

Hartmann C., Thüring E., Michels B.E., Pajonczyk D., Leußink S., Greune L., Brinkmann F., Glaesner-Ebnet M., Wardelmann E., Zobel T., Schmidt M.A., Gerke V., & Ebnet K. (2021). A novel adhesive complex at the base of intestinal microvilli.

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Immunoprecipitation Protocol for Protein Analysis

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WB experiments were performed as previously described^{41 (link)}. For the immunoprecipitation assay, cells were harvested 24 h after transfection, washed twice with cold phosphate buffered saline (PBS, Sigma-Aldrich) and lysed in RIPA buffer (Tris/HCl pH 8.0, 50 mM, NaCl 150 mM, SDS 0.1%, sodium deoxycholate 1%, Triton X-100 1%), supplemented with protease inhibitor cocktail (PIC) and phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich). Lysates were sonicated 2 × 10 sec and cleared, and proteins were quantified by Bio-Rad Protein assay (Bio-Rad, Hercules, California, USA). Lysates were incubated overnight at 4°C with the specific antibody, followed by 2 h incubation with protein G- or protein A-Sepharose beads (GE Healthcare Lifescience, Little Chalfont, UK). Immune complexes were washed four times in washing buffer and boiled in 2× sample buffer. The proteins were separated by electrophoresis on SDS-PAGE and detected using specific antibodies. Protein band intensities were quantified by densitometric analysis using Image J software.

Bellet M.M., Pieroni S., Castelli M., Piobbico D., Fallarino F., Romani L., Della-Fazia M.A, & Servillo G. (2020). HOPS/Tmub1 involvement in the NF-kB-mediated inflammatory response through the modulation of TRAF6. Cell Death & Disease, 11(10), 865.

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Immunoprecipitation of Protein Complexes

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Transfected COS7 cells were collected in immunoprecipitation (IP) buffer: 50 mM Tris-HCl (pH 8.0), 4.5 mM NaCl, 1% Nonidet P-40, with phosphatase inhibitors (Sigma), and protease inhibitors (Roche). Lysates were pre-cleared with sepharose beads for 30 minutes at 4°C. Protein-G or protein-A sepharose beads (GE Healthcare) were incubated with antibodies for 1 hour at 4°C. Beads were collected by centrifugation at 4000×rpm for 5 minutes. The beads bound to the antibodies were then incubated with the pre-cleared lysate overnight. Bead-protein complexes were washed 5 times with IP buffer and resuspended in SDS sample buffer containing dithiothreitol. The samples were separated by SDS-PAGE. Samples defined as “Input” were lysates not incubated with beads or antibody; the percentage refers to the amount of sample loaded onto the gel compared to the amount added to beads.

Burrell T.C., Divekar S.D., Weeber E.J, & Rebeck G.W. (2014). Fyn Tyrosine Kinase Increases Apolipoprotein E Receptor 2 Levels and Phosphorylation. PLoS ONE, 9(10), e110845.

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