293T or 293T cells expressing WT, K354R, or K354Q SAMHD1-GFP were not irradiated or irradiated with 10 Gy IR for 4 h. Cells were washed with PBS and cell lysates were prepared by resuspending in Buffer B (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 10% Glycerol, 10ug/mL BSA, 0.05% NP40, CHAPS 0.35%, protease inhibitors) and incubating on ice for 20 min. Lysates were clarified by centrifugation and protein concentration in the supernatant was determined by Bradford assay. Whole cell lysates (600 μg–1 mg) were precleared with 30 μL streptavidin-conjugated agarose beads (Millipore # 69203) for 1 h at 4 °C, where the beads had been pre-washed in Buffer A (10 mM Tris-HCl pH 7.5, 100 mM NaCl). Precleared lysates were incubated with 30 μL streptavidin-conjugated beads pre-bound to 40 pmol biotinylated DNA overnight at 4 °C. As a negative control, precleared lysate was also incubated with beads not prebound to biotinylated DNA. Biotinylated DNA used were single-stranded (ss), double-stranded (ds), 5′ overhang or 3′ overhang and sequences are as previously described (Fig. 5a ). Lysate-beads-biotinylated samples were washed with Buffer B four times, boiled in 5X SDS sample buffer and western blotted with SAMHD1 or GFP antibody.
Streptavidin conjugated agarose beads
Manufactured by Merck Group
Sourced in United States
Streptavidin-conjugated agarose beads are solid-phase supports composed of agarose beads with covalently attached streptavidin. Streptavidin is a tetrameric protein that binds strongly to the small molecule biotin. These beads can be used for the purification or immobilization of biotinylated molecules.
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Lab products found in correlation
S protein agarose beads, by Merck Group (1 mentions)
Set1a, by Cell Signaling Technology (1 mentions)
Anti flag m2 agarose bead, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti mouse and anti rabbit secondary antibodies, by Thermo Fisher Scientific (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Cd11a, by Abcam (1 mentions)
Smyd3, by Cell Signaling Technology (1 mentions)
Cd11b, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Amplex red cholesterol assay, by Thermo Fisher Scientific (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Immobilon p pvdf, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Mab11451, by R&D Systems (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
8 protocols using streptavidin conjugated agarose beads
1
SAMHD1 DNA Binding Assay
2
Purification and Analysis of SCML2 Interactome
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Purification of SFB triple-tagged protein (S, FLAG and SBP tags) was described previously [65] . To search for binding proteins of SCML2, we harvested 1 L cultures of 293 T cells stably expressing SFB-SCML2A, and washed the cell pellets with PBS. Cells were lysed with 30 mL ice-cold NETN300 buffer (0.5% NP-40, 50 mM Tris-HCl, pH 8.0, 2 mM EDTA and 300 mM NaCl). The soluble fraction was incubated with 0.5 mL streptavidinconjugated agarose beads (Millipore, 16-126). The beads were washed with NETN100 buffer (0.5% NP-40, 50 mM Tris-HCl, pH 8.0, 2 mM EDTA and 100 mM NaCl) three times. Associated proteins were eluted with 2 mM biotin (Sigma, B4501) in PBS, and subsequently incubated with 50 μL S-protein Agarose beads (Millipore, 69704). The bound proteins were eluted with SDS sample buffer and analyzed by 10% SDS-PAGE and mass spectrometry. Cells expressing empty vector were used as purification controls.
3
Validating miRNA-lncRNA Interactions
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To verify the putative miRNA‐lncRNA interaction, we used a biotinylated LBX2‐AS1 (Ribobio) as probe to enrich LBX2‐AS1‐interacting miRNAs from the cell lysate of the SKOV3 and OVCAR‐3 cells. Briefly, the cells were lysed using a harsh RIPA lysis buffer by pipetting, after which the cell lysate pre‐cleared by centrifugation was incubated with the biotinylated LBX2‐AS1 for 2 hours at 4 degree with gentle rotation, followed by incubation with streptavidin‐conjugated agarose beads (Sigma‐Aldrich) for 2 hours under the same condition. miRNAs associated with the beads were then subjected to qRT‐PCR as described above.
4
Mapping Methylation Sites in cGAS Protein
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The rabbit polyclonal anti-cGAS K491me antibody was produced by Boer Biotechnology (Chengdu, China). The mouse cGAS K491me peptide was synthesized and injected into rabbits. An affinity column with bound nonmodified peptide was used to collect and purify rabbit serum, excluding antibodies targeting nonmethylated cGAS. The antibody was then purified using an affinity column linked with bound cGAS K491me peptide. A rabbit polyclonal antibody recognizing human cGAS K506me was produced following the same protocol.
Antibodies recognizing cGAS, Tubulin, GST, His, SET1A, SET1B, SET7, MLL1, MLL3, γH2A.X and SMYD3 were obtained from Cell Signaling Technology. Antibodies recognizing RIOX2 were purchased from Atlas Antibodies. Antibodies recognizing RIOX1, ASH1L, PARP1, MLL2, SGF29, Timeless, NSD1, NSD2, H2A.X, CD11b, CD11a, CD34, Thy-1, and SMYD2 were purchased from Abcam. The antibody against Flag, anti-Flag M2 agarose beads, streptavidin-conjugated agarose beads, biotin, bovine serum albumin and poly(ADP-ribose) glycohydrolase (PARG) recombinant protein were purchased from Sigma. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies were purchased from Thermo Fisher Scientific. biotin (terminal)-PAR Polymer was purchased from Trevigen.
Antibodies recognizing cGAS, Tubulin, GST, His, SET1A, SET1B, SET7, MLL1, MLL3, γH2A.X and SMYD3 were obtained from Cell Signaling Technology. Antibodies recognizing RIOX2 were purchased from Atlas Antibodies. Antibodies recognizing RIOX1, ASH1L, PARP1, MLL2, SGF29, Timeless, NSD1, NSD2, H2A.X, CD11b, CD11a, CD34, Thy-1, and SMYD2 were purchased from Abcam. The antibody against Flag, anti-Flag M2 agarose beads, streptavidin-conjugated agarose beads, biotin, bovine serum albumin and poly(ADP-ribose) glycohydrolase (PARG) recombinant protein were purchased from Sigma. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies were purchased from Thermo Fisher Scientific. biotin (terminal)-PAR Polymer was purchased from Trevigen.
5
Immunoprecipitation and Quantification of ApoE
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Streptavidin-conjugated agarose beads (Sigma) were first incubated with biotin-conjugated goat anti-apoE antibody (K74180B, Meridian Life Science) for 2 h at room temperature with shaking. The unbound antibodies were removed by washing with TBS three times. The antibody-bound agarose beads were then mixed with brain lysates in TBS fractions (500 μg total protein) at 4°C overnight. The IP solution was centrifuged for 2 min at 1,000 g to collect agarose beads. The beads were washed with cold TBS three times and re-suspended with TBS/0.1% Triton X-100. The resulting suspension was subjected to Western blot analysis in which equal volumes of samples were loaded. Proteins were separated by 4-20% Mini-PROTEAN TGX Gel (Bio-Rad) and transferred to Immobilon-P PVDF (Millipore). The membranes were incubated with apoE antibody (K74180B, Meridian Life Science), followed by IRDye secondary antibody (LI-COR Biosciences). The results were visualized and quantified by Odyssey infrared imaging system (LI-COR Biosciences). The remaining suspension was used for the detection of apoE particle-associated cholesterol quantified by Amplex Red cholesterol assay (Invitrogen) according to the manufacturer’s protocol.
6
RAGE Antibody-RAGE Protein Interaction
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To assess the interaction between RAGE antibody (#Mab11451, R&D systems) and RAGE protein, coimmunoprecipitation (co-IP) was performed. Firstly, the RAGE antibody was labeled with a fluorescence material (FITC). HK-2 cells (1 × 106) were treated with FITC-labeled RAGE antibody (10 μg/ml) for 1 h at 4 °C, and then HK-2 cells were treated with AGE-BSA (10 μg/ml) for 24 hours. Next, the cells were harvested and lysed on ice for 30 min with lysis buffer, containing 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% TX-100, 50 mM Tris, pH 8.0, and protease inhibitors cocktail. Cell debris was removed by centrifugation at 14,000 rpm for 10 min at 4 °C. The supernatant of lysate was incubated with or without biotin-conjugated-RAGE polyclonal antibody (2 μg/ml, #bs-4999R-Biotin, Bioss) in the presence of streptavidin-conjugated agarose beads (#S1638, Sigma-Aldrich) at 4 °C overnight. The immunoprecipitates were washed 5 times with washing buffer (150 mM NaCl, 20 mM Tris, pH7.4, 0.1% TX-100) and detected the fluorescent signaling by using a ELISA reader.
7
Cell Surface Protein Biotinylation Assay
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The cell surface protein biotinylation assay was performed as described [33 (link)]. The arrest of endocytic traffic was attained by incubating cells with ice-cold PBS–CM (PBS pH 8.0 containing 1 mM CaCl2 and 1 mM MgCl2) for 5 min. Cell surface proteins were then labeled for 45 min on ice with sulfosuccinimidyl 3-[[2-(Biotinamido)ethyl] di-thio] propionate sodium salt (0.5 mg/mL, sc-212981, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Non-reacting biotin molecules were inactivated with quenching buffer (100 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glycine, 1% (w/v) BSA), and cells were lysed in pull-down buffer [50 mM Tris/HCl pH 7.5, 100 mM NaCl, 10% (v/v) glycerol, 1% (v/v) NP40, 0,1% (v/v) SDS supplemented with protease inhibitor cocktail composed of 1 mM PMSF, 1 mM 1,10-phenanthroline, 1 mM EGTA, 10 μM E64, and 10 μg/mL of each aprotinin, leupeptin and pepstatin A (all from Sigma-Aldrich, St. Louis, MO, USA)]. An aliquot of each cell lysate, representing input protein levels, was collected and the remaining lysate was incubated with streptavidin-conjugated agarose beads (Sigma-Aldrich, St. Louis, MO, USA). Captured proteins were solubilized 2 × in Laemmli buffer supplemented with 100 mM dithiothreitol (DTT)] and analyzed by western blotting.
8
Biotinylated LBX2-AS1 Enrichment
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To verify the putative miRNA-lncRNA interaction, we used a biotinylated LBX2-AS1 (Ribobio) as probe to enrich LBX2-AS1-interacting miRNAs from the cell lysate of the SKOV3 and OVCAR-3 cells. Brie y, the cells were lysed using a harsh RIPA lysis buffer by pipetting, after which the cell lysate pre-cleared by centrifugation was incubated with the biotinylated LBX2-AS1 for 2 hours at 4 degree with gentle rotation, followed by incubation with streptavidin-conjugated agarose beads (Sigma-Aldrich) for 2 hours under the same condition. miRNAs associated with the beads were then subject to qRT-PCR as described above.
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