Protein a agarose beads

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Protein A agarose beads are a type of affinity chromatography resin used for the purification of antibodies. They consist of Protein A, a bacterial protein that binds to the Fc region of immunoglobulins, immobilized on an agarose bead matrix. The beads provide a platform for the efficient capture and isolation of antibodies from complex samples such as cell culture supernatants or serum.

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Lab products found in correlation

Protease inhibitor cocktail, by Roche (3 mentions) Protein a agarose beads, by Beyotime (3 mentions) Magna rip rna binding protein immunoprecipitation kit, by Merck Group (2 mentions) Ripa buffer, by Santa Cruz Biotechnology (2 mentions) Protein g agarose bead, by Santa Cruz Biotechnology (2 mentions) Monoclonal antibody clone 11a50 b10, by BioLegend (2 mentions) Polyclonal anti aβ1 43, by Novus Biologicals (2 mentions) Polyclonal anti aβ1 43, by Santa Cruz Biotechnology (2 mentions) Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (2 mentions) Proteinase k, by Transgene (2 mentions) Anti flag, by Merck Group (2 mentions) Rneasy mini kit, by Qiagen (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Bis tris gel, by Thermo Fisher Scientific (1 mentions) Proteinase inhibitor, by Roche (1 mentions) Phosphatase inhibitor, by Roche (1 mentions) Tonitrocellulose membranes, by Merck Group (1 mentions) Protein g sepharose, by GE Healthcare (1 mentions) Fludarabine, by Merck Group (1 mentions) Diethyldithiocarbamate, by Merck Group (1 mentions)

Close spelling variants of this product

Protein A/G agarose beads Protein A/G PLUS-Agarose beads Protein A/G agarose bead slurry Protein A agarose Protein A beads Agarose beads Agarose

96 protocols using protein a agarose beads

Immunoprecipitation and Western Blot

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Cell lysates were prepared using the lysis buffer as described in western blot analysis. The lysates were incubated with primary antibodies suitable for immunoprecipitation overnight at 4°C. The mixtures were then incubated with Protein A-Agarose beads (Santa Cruz Biotechnology, Inc.) for 2 h, and the supernatant was collected by centrifugation at 1500 g. The Protein A-Agarose beads were washed three times with cold PBS and immunoprecipitates were dissolved in SDS and analyzed by western blot.

Kang J.H., Choi M.Y., Cui Y.H., Kaushik N., Uddin N., Yoo K.C., Kim M.J, & Lee S.J. (2017). Regulation of FBXO4-mediated ICAM-1 protein stability in metastatic breast cancer. Oncotarget, 8(47), 83100-83113.

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Immunoprecipitation of Renal NHERF1 and Tmem174

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Renal BBMVs of mice were lysed for 30 min at 4 °C in TNE lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, pH7.5), centrifuged for at 12,000×g, 10 min at 4 °C, and then supernatants were collected for immunoprecipitation. Immunoprecipitation samples were adjusted to 200 μg proteins/ ml in tubes, and anti-NHERF1 (LS-C46891, Lifespan Biosciences, Inc., Seattle, WA, USA) or anti- Tmem174 antibodies were added to tubes and rotated at 4 °C overnight. Next, protein A agarose beads (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were added to the tubes and rotated at 4 °C for 1 h. protein A agarose beads were centrifuged at 3000×g for 1 min at 4 °C and washed with TNE lysis buffer 4 times before removing the supernatant and eluting in SDS sample buffer. Loading samples were heated at 95 °C for 5 min and then analyzed by SDS-PAGE using antibodies against NHERF1, Tmem174, and NaPi2a.

Sasaki S., Shiozaki Y., Hanazaki A., Koike M., Tanifuji K., Uga M., Kawahara K., Kaneko I., Kawamoto Y., Wiriyasermkul P., Hasegawa T., Amizuka N., Miyamoto K.I., Nagamori S., Kanai Y, & Segawa H. (2022). Tmem174, a regulator of phosphate transporter prevents hyperphosphatemia. Scientific Reports, 12, 6353.

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Immunoprecipitation and Ubiquitin Detection

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Cells were lysed at 4°C in ice-cold immunoprecipitation assay buffer (Cell Signaling Technology, 9806) and cell lysates were cleared by brief centrifugation (13,000 g, 15 min). Concentrations of proteins in the supernatant were determined using the BCA assay. For immunoprecipitation assay, cell lysate was incubated with anti-Flag M2 beads or IP-proved antibodies overnight at 4 °C on a rotator, followed by the addition of protein A/G plus agarose to the reaction for 2 h at 4 °C. To detect ubiquitin conjugates, the lysates were denatured in lysis buffer containing 1% SDS to disrupt any protein-protein interactions, and then diluted ~10 fold prior to IP followed by immunoblot. Prior to immunoprecipitation, samples containing equal amounts of proteins were pre-cleared with protein A agarose beads (Santa Cruz Biotechnology, sc-2027; 4°C, 3 h), and subsequently incubated with various irrelevant IgG or specific antibodies in the presence of protein A agarose beads for 2 h or overnight at 4°C with gentle shaking. After five washes with lysis buffer supplemented with protease inhibitor mixture, complexes were released from the anti-Flag M2 beads by boiling for 5 min in 2x SDS-PAGE loading buffer.

Song X., Zhou Z., Li H., Xue Y., Lu X., Bahar I., Kepp O., Hung M.C., Kroemer G, & Wan Y. (2020). Pharmacological suppression of B7-H4 glycosylation restores antitumor immunity in immune-cold breast cancers. Cancer discovery, 10(12), 1872-1893.

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Immunoprecipitation and Western Blotting

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Lysates were prepared with lysis buffer as described in western blotting. After quantifying the protein levels, the lysates were incubated overnight with the primary antibody. The next day, protein A-agarose beads (Santa Cruz) were added to the sample tube and incubated for 2 h at 4 °C in an orbital shaker, and the protein A-agarose beads were washed three times with cold PBS and followed by Western blotting analysis.

Lim E.J., Kang J.H., Kim Y.J., Kim S, & Lee S.J. (2022). ICAM-1 promotes cancer progression by regulating SRC activity as an adapter protein in colorectal cancer. Cell Death & Disease, 13(4), 417.

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Immunoprecipitation and Western Blot

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Each cell lysate (500 μg), precleared with protein A–agarose beads (Santa Cruz Biotechnology), was incubated with 3 μg of anti-FLAG or anti-14-3-3τ antibody at 4°C overnight, immobilized on protein A–agarose beads, washed five times in lysis buffer, eluted by boiling, and subjected to SDS-PAGE and immunoblotting.

Lee T.G., Jeong E.H., Kim S.Y., Kim H.R., Kim H, & Kim C.H. (2017). Fhit, a tumor suppressor protein, induces autophagy via 14-3-3τ in non-small cell lung cancer cells. Oncotarget, 8(19), 31923-31937.

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Immunoprecipitation of α-klotho from mouse serum

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Mouse serum (125 μl) was mixed with 12.5 μl of 10% SDS, boiled at 100°C for 4 min, and cooled to room temperature. The sample was mixed with 0.4 ml of the TNE lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, pH 7.5) and centrifuged at 17,700 × g for 15 min at 4°C. The supernatant was collected and rotated at 4°C overnight with 1 μg anti-α-klotho antibody (R&D system, Minneapolis, MN). The next day, a 15-μl aliquot of protein A agarose beads (Santa Cruz Biotechnology, Inc., Dallas, TX) was added to the sample and rotated at 4°C for 1 h. protein A agarose beads were centrifuged at 3,000 × g for 1 min at 4°C and washed four times with the TNE lysis buffer. The agarose beads were removed from the supernatant, eluted in the SDS sample buffer, and heated at 95°C for 5 min. The sample was analyzed by immunoblotting as described above.

Koike M., Sato T., Shiozaki Y., Komiya A., Miura M., Higashi A., Ishikawa A., Takayanagi K., Uga M., Miyamoto K.I, & Segawa H. (2024). Involvement of α-klotho in growth hormone (GH) signaling. Journal of Clinical Biochemistry and Nutrition, 74(3), 221-229.

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Chromatin Immunoprecipitation for AR and SLIRP

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Cells were lysed in lysis buffer containing 50 mmol/L Tris-HCl, 0.1% NP40, 150 mmol/L NaCl, 10% glycerol, 2 mmol/L EDTA, plus proteinase inhibitor (Roche Diagnostic, Indianapolis, IN, USA) and phosphatase inhibitor (St. Louis, MO, USA). Immunoprecipitation was done by incubating the mixture of 500 μg protein lysis with 2 μg IgG and 50 μL protein A agarose beads (santa cruz biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. Immunoprecipitated fraction was resolved on 4–12% Bis-tris gel (Invitrogen, Carlsbad, CA, USA). For immunoblottting, antibodies were prepared at 1:1000 dilution unless specifically indicated. ChIP analysis was performed following the protocol described before^{10 (link)}. Briefly, an antibody against AR (Santa Cruz Technology, Santa Cruz, CA) or SLIRP (Abcam, Cambridge, MA, USA) was applied to immunoprecipitate DNA which is associated with AR and SLIRP. DNA was subjected to quantitative PCR using the primers and the probe targeting the distal ARE III enhancer sequence of the PSA gene or the primers and the probe targeting the distal enhancer of the hK2 gene as described previously^{10 (link)}.

De Silva D., Zhang Z., Liu Y., Parker J.S., Xu C., Cai L., Wang G.G., Earp H.S, & Whang Y.E. (2019). Interaction between androgen receptor and coregulator SLIRP is regulated by Ack1 tyrosine kinase and androgen. Scientific Reports, 9, 18637.

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Co-immunoprecipitation of AGGF1 and p53 Interactions

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Co-IP was carried out as described previously [10 (link),17 (link),35 (link)]. HCT116 cells were co-transfected with
pEGFP-P53 and pFLAG-AGGF1, pEGFP-P53 and wild type (WT) or different mutant
pCMV-C-HA-AGGF1 expression plasmids, or pEGFP-P53 and pEGFP-C1-AGGF1-FHA. The
transfected cells were lysed in lysis buffer (1% NP-40, 50 mM Tris-Hcl [pH 8.0],
150 mM NaCl, 10 mM NaF, 1 mM Na₃VO₄,
phenylmethane-sulfonylfluoride, and Roche EDTA-free protease inhibitors).
Cellular lysates were sonicated and centrifuged to remove insoluble materials,
and then incubated with protein-A agarose beads (Santa Cruz) at 4 °C for
1 h. The cleared lysates (1 ml) were divided into identical aliquots of 500
μl and incubated with 1 μg of the antibody for immunoprecipitation
(anti-EGFP, anti-HA, anti-FLAG or p53 and mouse or rabbit IgG as negative
control) at 4 °C overnight. Samples were incubated with protein G
sepharose (GE Healthcare) for 2–4 h, and washed. The bound proteins were
eluted with 2X SDS-PAGE buffer and separated by SDS-PAGE, transferred to
nitrocellulose membranes (Millipore) and probed with Western blotting antibodies
(anti--FLAG, anti-GFP or anti-p53).

Si W., Zhou B., Xie W., Li H., Li K., Li S., Deng W., Shi P., Yuan C., Ke T., Ren X., Tu X., Zeng X., Weigelt B., Rubin B.P., Chen Q., Xu C, & Kenneth Wang Q. (2020). Angiogenic factor AGGF1 acts as a tumor suppressor by modulating p53 post-transcriptional modifications and stability via MDM2. Cancer letters, 497, 28-40.

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Immunoprecipitation and Western Blotting of Bacterial Proteins

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Immunoprecipitations with the anti-FtsZ or anti-GFP antibodies were performed by mixing 1 mg of total protein from the lysates of the Spo0M:DsRed or the induced GFP:ZapA/Spo0M:DsRed strains with an anti-DsRed antibody for 3 h at 4°C. After this, 50 μL protein A agarose beads (sc-2001, Santa Cruz Biotechnology, Dallas, Texas, U.S.) were added to the mixture and incubated for 3 h at 4°C. The presence of FtsZ or GFP:ZapA was revealed by Western blotting with an anti-FtsZ or anti-GFP antibody. In the reverse order experiment, 1 mg of total protein from lysate of the Spo0M:DsRed or strain was mixed with an anti-FtsZ antibody for 3 h at 4°C. After this, 50 μL protein A agarose beads were added to the mixture and incubated for 3 h at 4°C. The presence of Spo0M:DsRed was revealed by Western blotting with an anti-DsRed antibody.

Vega-Cabrera L.A., Guerrero A., Rodríguez-Mejía J.L., Tabche M.L., Wood C.D., Gutiérrez-Rios R.M., Merino E, & Pardo-López L. (2017). Analysis of Spo0M function in Bacillus subtilis. PLoS ONE, 12(2), e0172737.

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Nuciferine Regulates YAP via AMPK

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PANC-1 cells were treated with nuciferine (50 μM) for 24 h. After treatment, the cells were washed and lysed for 15 min on ice and then centrifuged at 12000× g for 10 min and the soluble fraction was collected. Immunoprecipitation analysis AMPK was immunocaptured from total cell extracts using antibodies to AMPK crosslinked to protein A-agarose beads (Santa Cruz, CA, USA). The complexes were analyzed by Western blot and detected with antibody against YAP.

Zhou L., Wang Q., Zhang H., Li Y., Xie S, & Xu M. (2019). YAP Inhibition by Nuciferine via AMPK-Mediated Downregulation of HMGCR Sensitizes Pancreatic Cancer Cells to Gemcitabine. Biomolecules, 9(10), 620.

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