An immunoprecipitation kit from Proteintech (Wuhan, China) was used in this study. The main procedure was conducted according to the manufacturer's protocol. Briefly, cell lysates were immunoprecipitated with an antibody against USP22. Precipitated proteins (10 μg) were prepared for western blot analysis, and the detailed procedure has been described previously (Ling et al., 2014a ). Primary antibodies (described in the section 2.2. ) were incubated at 4 °C overnight. The bands were visualized by chemiluminescence, imaged using a ChemiDoc XRS, and analyzed using Image Lab (both from Bio‐Rad, Hercules, CA, USA).
Immunoprecipitation kit
Manufactured by Proteintech
Sourced in United States
The Immunoprecipitation Kit is a laboratory tool designed to isolate and purify specific proteins and their associated complexes from cell or tissue samples. The kit provides the necessary reagents and protocols to facilitate the immunoprecipitation process, which involves the use of antibodies to capture target proteins of interest.
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4 protocols using immunoprecipitation kit
1
Immunoprecipitation of USP22 Protein
2
SIRT1 Enzymatic Activity Assay
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SIRT1 enzymatic activity was measured using a commercial kit (ab156065; Abcam). According to the manufacturer's directions, fresh intestinal tissues were immunoprecipitated (Immunoprecipitation Kit; Proteintech, Chicago, IL, USA) with anti-SIRT1 antibody (Santa Cruz Biotechnology). Then, the reaction mixture containing fluoro-substrate peptide solution and protein A agarose beads was added, and NAD-dependent deacetylase activity was measured based on fluorescence intensity at 1-2 min intervals at Ex/Em = 350/460 nm in SpectraMax M5. The activity is presented as the relative value compared to the control group.
3
Protein Immunoprecipitation Protocol
4
Co-Immunoprecipitation Protocol for Protein Interactions
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Co-IP was performed using the Immunoprecipitation Kit (#PK10008, Proteintech, Rosemont, IL, USA) according to the manufacturer’s instructions. Briefly, protein samples were extracted using IP Lysis buffer supplemented with protease inhibitor. Protein samples were quantified by the BCA protein assay kit. A total of 2 μg of specific capture antibody and incubation buffer were added to 2 mg of protein samples and incubated at 4 °C for 24 h. Then, 50 μL of protein A/G beads were added to spin columns to capture the antigen-antibody complex. The antigen-antibody complex was washed with washing buffer and separated using 80 μl of elution buffer. Then, 10 μL of alkali neutralization buffer and 23 μL of 5 × loading buffer were added to the complex and analyzed by Western blot.
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