Affi prep protein a beads

Manufactured by Bio-Rad

Affi-Prep Protein A beads are an affinity chromatography media used for the purification of immunoglobulins and other Fc-containing proteins. The beads consist of Protein A, a bacterial protein that binds to the Fc region of antibodies, immobilized on a polymeric matrix support.

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Lab products found in correlation

Protease inhibitor cocktail, by Roche (2 mentions) Anti myc, by Merck Group (1 mentions) Anti actin, by Thermo Fisher Scientific (1 mentions) Anti histone h3 ps10, by Cell Signaling Technology (1 mentions) Anti smc1, by Fortis Life Sciences (1 mentions) Irdye 680rd goat anti mouse igg secondary antibody, by LI COR (1 mentions) Goat anti rabbit igg secondary antibody, by LI COR (1 mentions) Pakt s473, by Cell Signaling Technology (1 mentions) Microcystin lr, by Alexis Biochemicals (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Flag m2 bead, by Merck Group (1 mentions) Flag p53, by Addgene (1 mentions) Metafectene, by Biontex (1 mentions) Anti myc tag, by Cell Signaling Technology (1 mentions) Nonimmune rabbit igg, by Thermo Fisher Scientific (1 mentions) T9026, by Merck Group (1 mentions) T4026, by Merck Group (1 mentions) Las 3000, by Fujifilm (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Protein A beads (13 citations) Affi-Prep® Protein A (3 citations) Affi-prep (2 citations)

12 protocols using affi prep protein a beads

Antibody-Based Protein Analysis Protocol

Cited 2 times

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The following antibodies were used in this study: anti-centromere antibody (ACA or CREST-ImmunoVision, HCT-0100), anti-PP2A-Aα (Santa Cruz, Sc-6112), anti-Histone H3-pS10 (Cell signaling, 9706), anti-Smc1 (Bethyl, A300-055A), anti-SET (Bethyl, A302-261A), AKT (Cell signaling, 4691S), pAKT (S473) (Cell signaling, 4060S), anti-actin (Thermo Scientific, MA5-11869), anti-pHec1 (phospho Ser55, GTX70017, GeneTex), and anti-Myc (Millipore, 11667149001). Anti-Sororin is a gift from Susannah Rankin. Anti-Sgo1 and anti-GFP antibodies were made in-house as described previously (Liu et al., 2013b (link); Kim and Yu, 2015 (link)).
Antibody dilution for immunoblotting was often 1:1000 unless specified.
The secondary antibodies were purchased from Li-COR: IRDye 680RD goat anti-mouse IgG secondary antibody (926-68070) and goat anti-rabbit IgG secondary antibody (926-32211).
Harvested cells were collected and lysed with SDS sample buffer. After being 5-min boiled, lysates were resolved by SDS–PAGE and blotted with indicated antibodies.
For immunoprecipitation, anti-Myc or anti-GFP antibodies were coupled to Affi-Prep Protein A beads (Bio-Rad) at a concentration of 1 mg/ml⁻¹.

Yang L., Zhang Q., Niu T, & Liu H. (2021). SET levels contribute to cohesion fatigue. Molecular Biology of the Cell, 32(13), 1256-1266.

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Immunoprecipitation of Phosphoproteins

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Antibodies were conjugated to Affi-Prep Protein A beads (Bio-Rad Laboratories) at a concentration of 0.3 mg/ml. We lysed 1 × 10⁷ cells in 1 ml NP-40 lysis buffer (50 mM HEPES, pH 7.4, 200 mM KCl, 0.3% NP-40, 10% glycerol, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, and 10 µg/ml each of leupeptin, pepstatin, and chymostatin). To preserve phosphorylation, lysis buffer was supplemented with 1 mM NaVO4 and 0.5 µM microcystin LR (Alexis Biochemicals). Corresponding lysates were centrifuged, incubated with protein A beads coupled with preimmune rabbit IgG at 4 °C for 1 h, and then incubated with protein A beads coupled with specific antibodies at 4 °C overnight. The beads were washed 5x with lysis buffer, boiled in Laemmli sample buffer for 3 min and resolved via SDS–polyacrylamide gel electrophoresis (PAGE).

Song H., Kim E.H., Hong J., Gwon D., Kim J.W., Bae G.U, & Jang C.Y. (2023). Hornerin mediates phosphorylation of the polo-box domain in Plk1 by Chk1 to induce death in mitosis. Cell Death and Differentiation, 30(9), 2151-2166.

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Generating Antibodies for Immunoblotting and Immunoprecipitation

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The anti-Wapl antibody was generated against a C-terminal fragment of human Wapl (residues 601–1190) as described previously (Wu et al., 2012 (link)). Rabbit polyclonal antibodies against eGFP, human Sororin_91–252, and human Pds5B_1140–1310 were raised at Yenzym Antibodies with purified recombinant proteins as antigens. A list of commercial antibodies is provided in SI. For immunoblotting, the blots were incubated first with primary antibodies at 1 μg/ml and then with either fluorescently labeled or horseradish peroxidase-linked secondary antibodies. The blots were scanned with an Odyssey Infrared Imaging System (LI-COR) or developed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). For immunoprecipitation, antibodies were coupled to Affi-Prep Protein A beads (Bio-Rad) at a concentration of 1 mg/ml. Cleared cell lysates were incubated with antibody beads for 3 h at 4°C. Proteins bound to beads were dissolved in SDS sample buffer, separated by SDS-PAGE, and blotted with the appropriate antibodies. See SI for details.

Ouyang Z., Zheng G., Tomchick D.R., Luo X, & Yu H. (2016). Structural Basis and IP6 Requirement for Pds5-dependent Cohesin Dynamics. Molecular cell, 62(2), 248-259.

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Flag-tagged Protein Immunoprecipitation

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293T cells were transfected with the indicated constructs (Flag-PDCD5, Flag-p53 (Addgene #10838), Flag-Tip60) for 24 h with Metafectene (Biontex) according to the manufacturer′s instructions and subsequently treated as indicated. Cells were lysed in IP-Buffer16 (link) (300 mM NaCl, 50 mM Tris pH 8.0, 0.4% NP-40, 10 mM MgCl₂, 2.5 mM CaCl₂ and protease inhibitors (Roche)) and immunoprecipitated with Flag-M2 beads (Sigma) or PDCD5 antiserum coupled overnight to Affi-Prep Protein A beads (Biorad). Mouse IgG (Santa Cruz Biotechnology) or pre-immuneserum served as controls, respectively. After incubating the lysate with antibody-coupled beads for 1 h, beads were washed three times in lysis buffer, unless otherwise indicated. Beads were boiled in SDS loading buffer and supernatant loaded on SDS-Page gels.

Bock F.J., Tanzer M.C., Haschka M.D., Krumschnabel G., Sohm B., Goetsch K., Kofler R, & Villunger A. (2015). The p53 binding protein PDCD5 is not rate-limiting in DNA damage induced cell death. Scientific Reports, 5, 11268.

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Immunoprecipitation and Immunoblotting of APC/C

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Rabbit polyclonal antibodies used for the immunoprecipitation of APC/C were as follows: for Xenopus APC/C, antibody raised against 17-amino acid C-terminal peptide of Cdc27; for human APC/C, antibody raised against 14- amino acid C-terminal peptide of APC4. Both antibodies were purified by affinity chromatography with the corresponding antigens. Antibodies were bound to Affi-Prep-Protein A beads (Bio-Rad) at a concentration of 0.5 mg/mL packed beads. For control immunoprecipitation, nonimmune rabbit IgG (Invitrogen no. 31235) was bound to Protein A beads at a similar concentration. The following mouse monoclonal antibodies were used for immunoblotting: for Xenopus Cdc20, Abcam no. 18217, 1:500; for pT79 of Xenopus Cdc20, monoclonal antibody BT2 (generously provided by Dr. Julian Gannon, Cancer Research UK, Clare Hall Laboratories, South Mimms, UK, and described in ref. 19 (link)), 2 μg/mL; anti-Cdc27 (AF3.1) Santa Cruz no. 9972, 1:200; anti-Myc-tag, Cell Signaling no. 2276, 1:1,000.

Shevah-Sitry D., Miniowitz-Shemtov S., Teichner A., Kaisari S, & Hershko A. (2022). Role of phosphorylation of Cdc20 in the regulation of the action of APC/C in mitosis. Proceedings of the National Academy of Sciences of the United States of America, 119(35), e2210367119.

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In vitro IFT-A complex binding assay

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In vitro IFT-A complex binding assays were performed as described previously (Mukhopadhyay et al., 2010 (link)). In short, high-spin lysates from IFT140^LAP RPE cell pellets (∼500 µl of packed cell volume) were immunoprecipitated using anti-GFP antibody cross-linked to Affi-Prep Protein A beads (Bio-Rad). The washed beads were treated overnight with PrecissionS, and the digests were eluted in a total of ∼400 µl of LAP150^N buffer (50 mM Hepes, pH 7.4, 150 mM KCl, 1 mM EGTA, 1 mM MgCl₂, 10% glycerol, and 0.05% NP-40). 30 µl of freshly prepared PrecissionS eluates were added to ^MBPTULP3 or ^MBPTUB isoform b immobilized on packed amylose resin in the presence of recombinant ^HisTULP3 (1–183 aa; 21 µg) in a total volume of 100 µl LAP150^N buffer and incubated for 90 min by mixing at 4°C. Flowthrough after 90 min of binding was denatured with 5× sample buffer. After three washes in LAP150^N buffer, ^MBPTUB/TULP3-bound proteins were eluted in 45 µl of 2× sample buffer.

Badgandi H.B., Hwang S.H., Shimada I.S., Loriot E, & Mukhopadhyay S. (2017). Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins. The Journal of Cell Biology, 216(3), 743-760.

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Immunoprecipitation and Immunoblotting of Centromeric Proteins

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The commercial antibodies used in this study were: mouse anti–CENP-H (ab88593, Abcam), mouse anti–CENP-I (ab168778, Abcam), rabbit anti-CENP-K (sc-81831, Santa Cruz), rabbit anti-GFP ((in house made polyclonal antibody), mouse anti-myc (11667203001, Roche)), mouse anti–β-tubulin (T4026, Sigma-Aldrich), human CREST autoimmune sera (HCT-0100, ImmunoVision), mouse anti-α-tubulin (T9026; Sigma-Aldrich). For immunoblotting, antibodies were used at 1 μg/ml for purified and monoclonal antibodies or at 1: 1000 dilutions for crude sera.
For immunoprecipitation, anti-MYC or anti-GFP antibodies were coupled to Affi-Prep Protein A beads (Bio-Rad) at a concentration of 1 mg/ml. HeLa cells were lysed with the Lysis Buffer (25 mM Tris–HCl pH 7.5, 75 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 1 mM DTT, 0.5 μM okadaic acid, 5 mM NaF, 0.3 mM Na3VO4 10 mM β-glycerophosphate and 50 units/ml Turbo-nuclease). After 2 h incubation on ice and then 10-min incubation at 37°C, the lysate was cleared by centrifugation for 20 min at 4°C at 14 000 rpm. The supernatant was incubated with the antibody beads for 2 h at 4°C. The beads were washed four times with the lysis buffer. The proteins bound to the beads were dissolved in SDS sample buffer, separated by SDS-PAGE and blotted with the appropriate antibodies.

Hu L., Huang H., Hei M., Yang Y., Li S., Liu Y., Dou Z., Wu M., Li J., Wang G.Z., Yao X., Liu H., He X, & Tian W. (2018). Structural analysis of fungal CENP-H/I/K homologs reveals a conserved assembly mechanism underlying proper chromosome alignment. Nucleic Acids Research, 47(1), 468-479.

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Co-immunoprecipitation of ELKS Protein

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For co-immunoprecipitation, proteins were extracted from INS-1E cells in a Triton X-100 lysis buffer [20 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 1% Triton X-100, and a protease inhibitor cocktail (Roche)] with gentle rotation for 30 min at 4°C. All further steps were also executed at 4°C. The sample was centrifuged for 20 min at 16,100 g to obtain the supernatant, which was further incubated with Affi-prep Protein A beads (Bio-Rad) for preclearing for 30 min. The sample was centrifuged for 10 min at 16,100 g and the supernatant was further incubated with rabbit control serum or anti-ELKS antibody (Proteintech) for 1 h. After adding Affi-prep Protein A beads, the sample was further incubated for 1 h with rotation. The beads were washed three times before the proteins were eluted by boiling them in an SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE followed by western blotting for analysis.

Noordstra I., van den Berg C.M., Boot F.W., Katrukha E.A., Yu K.L., Tas R.P., Portegies S., Viergever B.J., de Graaff E., Hoogenraad C.C., de Koning E.J., Carlotti F., Kapitein L.C, & Akhmanova A. (2022). Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells. Journal of Cell Science, 135(3), jcs259430.

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Monitoring Cdc20 Binding to APC/C

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To examine levels of Cdc20 associated with APC/C, the APC/C was immunopurified from mitotic Xenopus egg extract. For 100 μl extract, 2 μg of anti-Cdc27 antibody (AF3.1, Santa Cruz Biotechnology) was cross-linked to 5 μl of Affiprep Protein A beads (156-0006, Bio-Rad) and incubated for 1 h at 4 °C. Apcin, TAME or DMSO was mixed with extract upon addition to anti-Cdc27-Affiprep Protein A beads in the presence or absence of cycB1-NT containing a HA-tag at N-terminus and his tag at C-terminus, as previously described³⁴. Following incubation with extract, beads were washed quickly three times with 20-fold volume of XB (10 mM potassium HEPES, pH 7.7, 100 mM KCl, 0.1 mM CaCl₂, 1 mM MgCl₂) and combined with SDS sample buffer. For analysis of Cdc20 binding to APC/C, samples were processed for SDS-PAGE and immunoblotting against Cdc20 and APC/C subunit Cdc27. Chemiluminescence was imaged on a Fuji LAS 3000 with Image Reader LAS-3000 software. Levels of Cdc20 were quantified using Image J and data was normalized to respective Cdc27 levels.

Sackton K.L., Dimova N., Zeng X., Tian W., Zhang M., Sackton T.B., Meaders J., Pfaff K.L., Sigoillot F., Yu H., Luo X, & King R.W. (2014). Synergistic Blockade of Mitotic Exit by Two Chemical Inhibitors of the APC/C. Nature, 514(7524), 646-649.

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Immunoprecipitation and Activity Assay

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Whole cell lysates or sucrose gradient fractions containing a protease inhibitor cocktail (Roche) were incubated with antibody for two hours at 4°C. Pre-washed affiprep Protein A beads (BioRad) were added and incubated for one hour at 4°C with the antibody/antigen complex. Beads were washed with 50mM Tris pH 8.0, 50mM NaCl, 1mM EGTA, 1mM MgCl2. SILAC samples were washed with 50mM Tris pH 8.0, 150mM NaCl, 1mM EGTA, 1mM MgCl2. For SDS-PAGE analysis, proteins were eluted from the beads by boiling in sample buffer, and immunoblots were performed using the Licor Quick Western Dectection Kit. Activity assays were performed directly on beads.

Krukenberg K.A., Jiang R., Steen J.A, & Mitchison T.J. (2014). Basal activity of a PARP1-NuA4 complex varies dramatically across cancer cell lines. Cell reports, 8(6), 1808-1818.

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