Cells were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich; EMD Millipore) as previously described (22 (link)–24 (link)). Cells were centrifuged at 4°C for 10 min at 16,000 × g. Protein concentrations were determined by the Bradford assay (25 (link)). Aliquots containing 20 µg of total protein were separated by 10% sodium dodecyl-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blots were probed with primary antibodies against USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (1:5,000, mouse monoclonal; A5316; Sigma-Aldrich; EMD Millipore). Appropriate secondary antibodies (1:3,000; rabbit anti-goat; ab6741; 1:5,000; rabbit anti-mouse; ab97046; Abcam, Cambridge, MA, USA) conjugated to horseradish peroxidase and enhanced chemiluminescence (GE Healthcare Life Sciences, Chalfont, UK) were used to detect the bound primary antibodies. Co-IP was performed with cell lysate (500 µg) incubated with USP21 (1:1,000; goat polyclonal; sc-79305; Santa Cruz Biotechnology, Inc.), relA (1:1,000; rabbit polyclonal; sc-372; Santa Cruz Biotechnology, Inc.) or non-specific-IgG antibodies (1:1,000; rabbit IgG, monoclonal; ab172730; Abcam) using µMACS™ Protein A/G MicroBeads and MACS® Separation Columns according to the manufacturer's protocol (Miltenyi Biotec, Auburn, USA).
Macs protein a g microbeads
Manufactured by Miltenyi Biotec
Sourced in United States, Germany
The µMACS™ Protein A/G MicroBeads are a laboratory product designed for the rapid and efficient purification of antibodies and other proteins from biological samples. These magnetic beads are coated with a combination of Protein A and Protein G, which have a high affinity for the Fc region of antibodies. The beads can be used in a variety of applications, including immunoprecipitation, affinity purification, and protein isolation.
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Lab products found in correlation
Macs separation column, by Miltenyi Biotec (1 mentions)
A5316, by Merck Group (1 mentions)
Ab6741, by Abcam (1 mentions)
Ab97046, by Abcam (1 mentions)
Ab172730, by Abcam (1 mentions)
Sc 372, by Santa Cruz Biotechnology (1 mentions)
Sc 79305, by Santa Cruz Biotechnology (1 mentions)
Sypro ruby protein gel stain, by Thermo Fisher Scientific (1 mentions)
2 protocols using macs protein a g microbeads
1
Protein Extraction and Immunoblotting for USP21 in Cell Lysates
2
Immunoprecipitation of Antigen-Antibody Complex
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The HMOS-P cell line was lysed using the ULTRARIPA® Kit (BioDynamics Laboratory Inc., Tokyo, Japan) to prepare cytoplasmic and cell membrane lysates. The lysate was individually incubated with antibody number 12, and an anti-rabbit cp3 polyclonal antibody was added as the secondary antibody. The antigen–antibody conjugates were adsorbed by incubating with µMACS™ Protein A/G MicroBeads (Miltenyi Biotec BV & Co. KG, Gladbach, Germany). This reaction solution was then incubated with a MultiMACS™ Protein A/G kit (Miltenyi Biotec BV & Co. KG), and the antigen–antibody complex was washed and concentrated by immunoprecipitation using magnetic beads. The antigen–antibody complex was then cleaved by enzymes, released from the magnetic beads, and eluted from the column.
The cell lysate, the negative control (with only the antibody), and the antigen–antibody complex incubated with the cell lysate were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with SYPRO® Ruby Protein Gel Stain (Thermo Fisher Scientific K.K.), and bands were detected using FLA3000G (FUJIFILM Healthcare Laboratory Co., Ltd., Tokyo, Japan). Finally, the Coomassie-stained gels were used to visualize the regions where the antigen–antibody complex was detected.
The cell lysate, the negative control (with only the antibody), and the antigen–antibody complex incubated with the cell lysate were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with SYPRO® Ruby Protein Gel Stain (Thermo Fisher Scientific K.K.), and bands were detected using FLA3000G (FUJIFILM Healthcare Laboratory Co., Ltd., Tokyo, Japan). Finally, the Coomassie-stained gels were used to visualize the regions where the antigen–antibody complex was detected.
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