Cells, pretreated with 10uM MG132, 50uM of Leupeptin (Fisher Scientific) and 0.3mMprimaquine (Sigma) where indicated, were labeled for 30min on ice with Sulfo-NHS-S-S-biotin (Thermo Fisher Sci.) in PBS; followed by incubation at 37°C to allow internalization and recycling of the labeled surface molecules for times as indicated on the figures. At each time point, the residual surface biotin was removed by 10 min incubation with 200 mM MESNA (Sigma) three times. Post-treatment cells were homogenized in PTY buffer [1 ] and biotinylated molecules were pulled down with streptavidin-conjugated agarose (EMD Millipore), loaded on a gel, transferred onto PVDF membrane to perform western blot analysis with anti-integrin β1 antibody.
Streptavidin conjugated agarose
Manufactured by Merck Group
Sourced in United States
Streptavidin-conjugated agarose is a laboratory product used for affinity purification and immobilization of biotinylated proteins, nucleic acids, and other biomolecules. It consists of streptavidin, a protein with a high affinity for biotin, covalently bound to an agarose matrix. This product facilitates the selective capture and separation of biotinylated target molecules from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Leupeptin, by Thermo Fisher Scientific (3 mentions)
Mesna, by Merck Group (3 mentions)
Sulfo nhs ss biotin, by Thermo Fisher Scientific (3 mentions)
Mg132, by Thermo Fisher Scientific (2 mentions)
S nitrosylated protein detection assay kit, by Cayman Chemical (1 mentions)
Nuclear extract kit, by Active Motif (1 mentions)
6 protocols using streptavidin conjugated agarose
1
Integrin β1 recycling dynamics
2
Detection and Purification of S-Nitrosylated Proteins
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S-nitrosylated proteins were purified by the S-Nitrosylated Protein Detection Assay Kit (Cayman, Ann Arbor, MI, USA) and streptavidin-conjugated agarose (Merck). The assay employs a biotin switch method to biotinylate the S-nitrosylated proteins. Briefly, the free thiol groups of cell lysates (20×106 cells) were blocked by the blocking reagent of the kit. The S-nitrosothiols of proteins were then reduced and covalently labeled with maleimide-biotin. The biotin-labeled proteins were resuspended in 500 μL PBS and were purified by incubation with 25 μL of streptavidin-conjugated agarose at 4°C for 3 h. The purified S-nitrosylated proteins were resolved by SDS-PAGE and were visualized by silver staining or western blot as described above. Biotin conjugated myelin basic protein (ab792; Abcam) was added as a loading control of the protein purification process and was detected by polyclonal antibody against myelin basic protein (ab28541; Abcam).
3
Nuclear Protein Extraction and Analysis
4
Biotin-labeled Peptide Pull-down Assay
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Biotin-labeled synthetic peptide corresponding to helix in dmATL C-terminal tail was described previously (Liu et al., 2012 (link)). Peptides were bounded to streptavidin-conjugated agarose (Sigma), and then incubated with purified dmLnp-NT-Flag-GST for 40 min at 4 °C. The agarose was then washed with buffer A four times. Precipitated dmLnp-NT were eluted by 2× SDS-PAGE sample buffer and detected by Western blot. Streptavidin and imported peptides were measured by coomassie blue staining.
5
Biotinylation and Internalization Assay
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Cells pretreated with 10μM MG132 and 50μM Leupeptin (FisherScientific) were labeled at 4°C with Sulfo-NHS-S-S-biotin (ThermoFisher Scientific) in PBS, followed by incubation at 37°C for internalization of surface molecules. Residual surface biotinylation was removed by incubation with 200mM MESNA (Sigma) for 30 minutes. Cells were homogenized in PTY buffer (62 (link)), and biotinylated molecules were pulled down with streptavidin-conjugated agarose (Millipore) and used for western blotting.
6
Biotinylation and Internalization Assay
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