Protein a magnetic beads

Manufactured by Bio-Rad

Sourced in United States

Protein A magnetic beads are a type of laboratory equipment used for the purification and isolation of antibodies. They consist of superparamagnetic beads coated with protein A, a bacterial protein that binds to the Fc region of immunoglobulins. The magnetic properties of the beads allow for easy separation and washing of the bound antibodies using a magnetic field.

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Lab products found in correlation

Cp02 pulverizer, by Covaris (2 mentions) Complete mini protease inhibitor cocktail, by Roche (2 mentions) Protein g magnetic beads, by Bio-Rad (2 mentions) Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions) Lysis buffer, by Cell Signaling Technology (1 mentions) Rabbit igg, by Cell Signaling Technology (1 mentions) Ab2410, by Abcam (1 mentions) Pcr dna purification columns, by Qiagen (1 mentions) Cpth2, by Merck Group (1 mentions) Flag antibody, by Merck Group (1 mentions) Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Mouse igg, by Cell Signaling Technology (1 mentions) Anti ha tag antibody, by Santa Cruz Biotechnology (1 mentions) Odyssey infrared imager, by LI COR (1 mentions) Irdye conjugated secondary antibody, by LI COR (1 mentions) Protease inhibitors cocktail, by Merck Group (1 mentions) Glass bead, by Merck Group (1 mentions) Rabbit anti histone h3, by Cell Signaling Technology (1 mentions) Anti myc, by Roche (1 mentions)

26 protocols using protein a magnetic beads

Immunoprecipitation of Protein Phosphatase 1

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Immunoprecipitation of PP1 was carried out as described earlier.⁴ Briefly cells were lysed with lysis buffer (20mM Tris/Cl pH 8, 150mM NaCl, 0.5mM EDTA, 0.5% Triton X-100, 1mM PMSF and Protease inhibitor cocktail) and incubated for 2 hours with protein A magnetic beads (Biorad) bound to PP1 antibody. Beads were then washed 3 times with the same lysis buffer and proteins were eluted by boiling in 1X laemmli buffer.

Roy S., Esmaeilniakooshkghazi A., Patnaik S., Wang Y., George S.P., Ahrorov A., Hou J.K., Herron A.J., Sesaki H, & Khurana S. (2017). Villin-1 and Gelsolin Regulate Changes in Actin Dynamics That Affect Cell Survival Signaling Pathways and Intestinal Inflammation. Gastroenterology, 154(5), 1405-1420.e2.

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Co-Immunoprecipitation of Protein Complexes

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For co-immunoprecipitation, cell extracts composed of 5.0×10⁷ cells were prepared by solubilization in 1ml cell lysis buffer (0.5% NP-40, 10 mM NaCl, 10 mM Tris-HCl, pH 8.0, 0.3M Sucrose, 3 mM MgCl₂, phosphatase and protease inhibitors cocktail) for 15 min at 4°C. Centrifuge the lysate at 10,000 g for 10 min at 4°C, and the cell extract was immunoprecipitated with 7.5 μg antibodies against Flag and incubated with 50 μL of protein A Magnetic Beads (Bio-Rad, Hercules, CA, USA) overnight at 4 °C by continuous inversion. Immunocomplexes were pelleted and washed three times with PBST. The precipitated immunocomplexes were boiled in 2×Laemmli buffer.

Jiang X., Wu Z., Lu X., Zhang X., Yu Q., Gan Y., Wu B., Xu Y., Zheng W., Zhang L., Xu F., Ma A., Gan X., Huang S., Yu X., Huang W, & Xu R. (2017). Activation of CaMKIIγ potentiates T-cell acute lymphoblastic leukemia leukemogenesis via phosphorylating FOXO3a. Oncotarget, 8(43), 75050-75064.

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Immunoprecipitation of Cardiac Proteins from Mice

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30 μg of LV transmural sample from 8-week-old WT and KO mice were used for protein extraction and quantification was done using a BCA assay kit. Protein A magnetic beads (Bio-Rad, catalog#161-4011) were washed thrice with 1× PBST. 4 mg ml⁻¹ of lysate was precleared by rotating with beads for 30 min at 4 °C and 10% of the precleared lysate was saved as input. 5 μg of antibody (Table 1) was used for conjugation with the beads by rotating at room temperature for 10 min, followed by washing twice with 1× PBST and once with the lysis buffer (Cell Signaling, catalog #9806). The precleared lysate and antibody were kept for rotation at room temperature for 1 h. The magnet was then washed thrice with 1× PBST, magnetized and the elute was boiled at 70 °C for 10 min with 40 μl of 1× Laemmli buffer (10% β-mercaptoethanol) and used for western blot.

Shankar T.S., Ramadurai D.K., Steinhorst K., Sommakia S., Badolia R., Thodou Krokidi A., Calder D., Navankasattusas S., Sander P., Kwon O.S., Aravamudhan A., Ling J., Dendorfer A., Xie C., Kwon O., Cheng E.H., Whitehead K.J., Gudermann T., Richardson R.S., Sachse F.B., Schredelseker J., Spitzer K.W., Chaudhuri D, & Drakos S.G. (2021). Cardiac-specific deletion of voltage dependent anion channel 2 leads to dilated cardiomyopathy by altering calcium homeostasis. Nature Communications, 12, 4583.

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Immunoprecipitation of GFP-tagged Proteins

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For immunoprecipitation using ALFA-tagged nanobodies (NbVHH05-ALFA and Nb127D01-ALFA), His-tag purified nanobodies from bacteria were incubated with ALFA Selector ST resin (Nanotag Biotechnologies, N1511) at room temperature for 1 hr. The resin was washed with Pierce IP lysis buffer (Thermo Scientific, 87787) (3×) and incubated with S2 cell culture media containing secreted GFP proteins with 3xVHH05-tag and 3x127D01 tag at 4°C for 1 hr. After washing in IP lysis buffer (4×), proteins were eluted in 2× sample buffer.
For immunoprecipitation using hIgG-formatted nanobody (Nb127D01-hIgG), Protein A magnetic beads (Bio-Rad, 1614013) were incubated with S2 cell culture media containing Nb127D01-hIgG at room temperature for 1 hr. After washing in IP lysis buffer (3×), beads were incubated with the conditioned media containing secreted GFP proteins 3x127D01 tag at 4°C for 1 hr. After washing in IP lysis buffer (4×), proteins were eluted in 2× sample buffer.

Xu J., Kim A.R., Cheloha R.W., Fischer F.A., Li J.S., Feng Y., Stoneburner E., Binari R., Mohr S.E., Zirin J., Ploegh H.L, & Perrimon N. (2022). Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies. eLife, 11, e74326.

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Immunoprecipitation of PSD-95 from Synaptic Fractions

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For the immunoprecipitation (IP) experiment, the procedure for isolating PSDs was performed as described above for the characterization of protein marker distribution in PSDs (Fig. 1a). We adapted our IP strategy based on an affinity-based approach in a previous study [56 (link)]. Briefly, Protein A magnetic beads (Surebeads 161–4023, BioRad) were washed two times with phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH 7.4). Next, washed beads were incubated with either polyclonal PSD-95 antibody (ab18258) or rabbit IgG (#2729, Cell Signaling Technology) at 1:200 dilution in PBS for 1 h at ambient temperature. Beads with α-PSD-95 or rabbit IgG bound were then washed with Tris-buffered saline (TBS; 50 mM Trizma base, 150 mM NaCl, pH 7.6) with 0.1% Tween 20 three times for 5 min. Pre-sonicated P4 fractions were incubated with either PSD-95 antibody-coated beads or rabbit-IgG-coated beads for 2 h at 4 °C. Next, beads were collected by magnetic rack and washed three times for 3 min in TBS with 0.1% Tween 20. Elution was performed in RIPA buffer with sample buffer added (3X; 187.5 mM Trizma base, 6% SDS, 30% glycerol, 0.03% bromophenol blue, 15% β-mercaptoethanol, pH 7.6) with a 5 min incubation at 95 °C.

Xiao S., McKeever P.M., Lau A, & Robertson J. (2019). Synaptic localization of C9orf72 regulates post-synaptic glutamate receptor 1 levels. Acta Neuropathologica Communications, 7, 161.

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Immunoprecipitation and Immunoblotting of Protein Complexes

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Cells were harvested and whole cell lysates were prepared in RIPA lysis buffer as described (7 (link)) or in MIB lysis buffer (10 (link)) supplemented with 1x protease inhibitors and 1x phosphatase inhibitors (Roche) by sonication for 2 minutes. To make ER+ PDX tumor lysates, frozen PDX tumors were cryopulverized with a Covaris CP02 Pulverizer and then protein was extracted in MIB lysis buffer with sonication. Protein concentration determination and SDS-PAGE (20 μg protein per lane) were performed as described (7 (link)). Immunoblotting of nitrocellulose membranes was performed as described (7 (link)). Primary and HRP-conjugated secondary antibodies employed are listed in the Supplementary information.
Immunoprecipitation was performed as described (7 (link)), using 2 mg of lysates from hormone-deprived T47D cells with or without E2 treatment (100 nM for 45 minutes). Lysates were incubated with 2 μg anti-HA tag antibody (Santa Cruz Biotechnology Cat# sc-7392, RRID:AB_627809) or mouse IgG (Cell Signaling Technology Cat# 61656, RRID:AB_2799613) control, followed by capture of antibody-antigen complexes with protein A magnetic beads (Bio-Rad, cat# 1614013) as described (7 (link)). Immunoprecipitated proteins, as well as 20 μg of whole cell lysates (1% inputs), were analyzed by immunoblotting.

Gou X., Anurag M., Lei J.T., Kim B.J., Singh P., Seker S., Fandino D., Han A., Rehman S., Hu J., Korchina V., Doddapaneni H., Dobrolecki L.E., Mitsiades N., Lewis M.T., Welm A.L., Li S., Lee A.V., Robinson D.R., Foulds C.E, & Ellis M.J. (2021). Transcriptional Reprogramming Differentiates Active from Inactive ESR1 Fusions in Endocrine Therapy-Refractory Metastatic Breast Cancer. Cancer Research, 81(24), 6259-6272.

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Immunoprecipitation of CB1 Receptor

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Following tetracycline induction, CB1-CHO cells were washed with ice-cold PBS and suspended in IP buffer containing 50 mM tris-HCl, 120 mM NaCl, 0.5% NP-40, 0.2% n-dodecyl-β-d-maltopyranoside, 5% glycerol, and 1× protease inhibitor cocktail (pH 7.5). The lysate was centrifuged, and the resulting supernatant was incubated with the rabbit anti-CB1 (93815, Cell Signaling Technology) antibody for 20 min at 4°C. Immunocomplex was incubated with Protein A Magnetic beads (Bio-Rad, USA) overnight on a rotating wheel at 4°C. The beads was then washed five times in wash buffer containing 20 mM tris-HCl, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40 (pH 8.0). The immunoprecipitates were mixed with the loading buffer and resolved by SDS–polyacrylamide gel electrophoresis (PAGE). Western blots were performed with relevant antibodies. Rabbit IgG was used as a negative control.

Li S., Luo H., Lou R., Tian C., Miao C., Xia L., Pan C., Duan X., Dang T., Li H., Fan C., Tang P., Zhang Z., Liu Y., Li Y., Xu F., Zhang Y., Zhong G., Hu J, & Shui W. (2021). Multiregional profiling of the brain transmembrane proteome uncovers novel regulators of depression. Science Advances, 7(30), eabf0634.

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Chromatin Immunoprecipitation and Quantitative Analyses

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Chromatin immunoprecipitation (ChIP), quantitative RT-PCR, and ChIP-qPCR were performed as described earlier (13 (link),29 (link),30 (link)). Briefly, cells were either transfected, infected or treated with various inhibitors (R)-9b; CPTH2 (Sigma, Cat#C9873)], fixed in formaldehyde, lysed and ChIP was performed with antibodies recognizing AR (SCBT, sc-7305; RRID:AB_626671), GCN5 (SCBT, SC-365321; RRID:AB_10846182), H3K14ac (CST, 7627S; RRID:AB_10839410), H3K27me3 (CST, 9733S; RRID:AB_2616029), NCOR1 (Bethyl Laboratories, Cat#A301-145A; RRID:AB_873085), EZH2 (CST, 5246S; RRID:AB_10694683), or IgG (Abcam, ab2410; RRID:AB_303052), immobilized on Protein A magnetic beads (Biorad, Cat# 161-4013). The complexes were washed with ChIP buffers, eluted with elution buffer (Active Motif, Cat#53008), and subjected to reverse crosslinking, followed by RNase and proteinase K treatments. A part of soluble chromatin was processed separately at the same time and designated as input DNA. Treated ChIP DNA and input DNAs were purified using PCR-DNA purification columns (Qiagen, Cat#28106). The amount of immunoprecipitated DNA was determined by real-time PCR.

Ghildiyal R., Sawant M., Renganathan A., Mahajan K., Kim E.H., Luo J., Dang H.X., Maher C.A., Feng F.Y, & Mahajan N.P. (2021). Loss of long non-coding RNA NXTAR in prostate cancer augments androgen receptor expression and enzalutamide resistance. Cancer research, 82(1), 155-168.

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Immunoprecipitation of Protein Complexes

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20 mg portions of tissues were lysed in 600 μL lysis buffer with HEPES 40 mM pH 7.5, NaCl 120 mM, EDTA 1 mM, Na-pyrophosphate 10 mM, glycerophosphate 10 mM, NaF 50 mM and 0.3 % CHAPS. 2 μg antibody was mixed with pre-washed protein A magnetic beads (Biorad 1614013) and rotated at 4°C for 2 hrs. 300 μL tissue lysates were incubated with the antibody at 4°C overnight. Proteins were eluted with 40 μL 1X Laemmli buffer (Biorad 1610747) at 70°C for 10 min.

Shi X., Endicott S.J, & Miller R.A. (2022). Regulation of mTOR complexes in long-lived growth hormone receptor knockout and Snell dwarf mice. Aging (Albany NY), 14(6), 2442-2461.

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CATSPER1 Protein Immunoprecipitation

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Mouse testes were homogenized in 0.32M sucrose and cell debris and nuclei were removed by centrifugation at 1,000 × g, 4°C for 20 min. Supernatant was centrifuged at 100,000 × g, 4°C for 60 min to collect membrane microsome. The microsome fraction was solubilized in 1% Triton X-100 in PBS (10 mg microsome per ml) with protease inhibitor cocktail (complete mini, Roche) by rocking at 4°C for 60 min. The lysate was cleared by centrifugation at 18,000 × g, 4°C for 30 min to obtain solubilized proteins from the supernatant. The supernatant was immunoprecipitated with anti-CATSPER1 antibody and Protein A magnetic beads (BIO-RAD) with the same method for recombinant protein co-IP. Proteins were detected with corresponding rabbit raised primary antibodies and horseradish peroxidase-conjugated goat-anti-rabbit secondary antibody.

Huang X., Miyata H., Wang H., Mori G., Iida-Norita R., Ikawa M., Percudani R, & Chung J.J. (2023). A CUG-initiated CATSPERθ functions in the CatSper channel assembly and serves as a checkpoint for flagellar trafficking. bioRxiv.

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