THP-1 macrophages were lysed with IP lysate and protein samples mixed with Sirt3 or ATG5 antibodies (2 μg) and 20 μL of 50% protein A/G-agarose bead slurry (Pierce Biotechnology, Rockford, IL, USA) overnight at 4 °C with gentle rotation. Immune complexes were washed 5 times with lysis buffer [50 mmol/l Tris (pH 7.4), 1% Triton X-100, 0.5% Nonidet P-40, 150 mmol/l NaCl, protease inhibitor (Calbiochem), and 10% (vol/vol) glycerol]. Samples were boiled in 2× sample buffer and then subjected to SDS-PAGE. The specificity of antibodies used for immunoprecipitation was routinely validated by running negative controls using non-immune IgG under the same conditions. For analysis of acetylated ATG5 in THP-1 cells, 1 mg of protein lysate was directly immunoprecipitated with an acetyl-lysine antibody and subjected to western blotting for Sirt3 and ATG5.
Protein a g agarose bead slurry
Manufactured by Thermo Fisher Scientific
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Protein A/G agarose bead slurry is a laboratory reagent composed of agarose beads conjugated with Protein A and Protein G. It is commonly used in affinity chromatography for the purification and isolation of immunoglobulins and other proteins that bind to Protein A or Protein G.
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5 protocols using protein a g agarose bead slurry
1
Analysis of Sirt3 and ATG5 Acetylation in THP-1 Macrophages
2
Immunoprecipitation of SIRT1, YAP, and Acetylation
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For immunoprecipitation, HEK293T cell lysates were precleared and incubated with 2 μg of capture antibody and 20 μl of 50% protein A/G-agarose bead slurry (Pierce Biotechnology, Rockford, IL, USA) overnight at 4 °C with gentle rotation. Immunoprecipitates were washed three times with modified RIPA buffer and boiled in 2 × Laemmli buffer, then subjected to SDS-PAGE electrophoresis and detection with specific antibodies against SIRT1, YAP, FLAG, or acetyl-lysine (AcK). The beads were washed. The specificity of antibodies used for immunoprecipitation was routinely validated by running negative controls using non-immune IgG using the same conditions as in formal experiments.
3
Co-immunoprecipitation of mGlu5 and Homer
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Hippocampi were homogenized at 4°C in co-immunoprecipitation buffer (50 mM Tris, pH 7.4, 120 mM NaCl, 0.5% Igepal, 1 mM EDTA, 1 mM EGTA), and 1 mg of total proteins were tumbled overnight at 4°C with 5 μg of anti pan-Homer antibodies (Santa Cruz Biotechnology, D-3). Protein A/G agarose bead slurry (Thermo Fisher Scientific) was added for two additional hours, and the beads were then washed with co-immunoprecipitation buffer. Western blotting was performed with anti-mGlu5 and anti pan-Homer antibodies.
4
Characterization of Homer-mGluR5 Complexes
5
NLRP3 Protein Immunoprecipitation Protocol
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Protein was extracted with lysis buffer containing 0.1% Triton X-100, supplemented with 7.5 mM MgCl2 and lysates were cleared (17,000 × g, 4 °C, 20 minutes). 20 μl of Protein A/G agarose bead slurry (Thermo Scientific, 20421) were added to 25 μl of anti-NLRP3 antibody (CST, 15101) for 30 minutes at 25 °C. Lysates containing 1 mg of total protein were incubated with the bead-antibody conjugate at 4 °C under rotary agitation for 6 hours. Samples were centrifuged (10 sec, 2,000 × g), the supernatant (labelled post-IP) was separated from the beads and kept for validation of successful NLRP3 depletion form the sample. Beads were washed with lysis buffer 5 times, then washed with lysis buffer containing 0.5 M NaCl, then finally washed with normal lysis buffer. The antigen-antibody complex was eluted by heating at 95 °C in SDS-containing loading buffer (Thermo Fisher, NP0007) for 10 minutes. Immunoprecipitation was verified by Western blotting.
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