Competition experiments were performed in 50 mM phosphate buffer (pH 8) supplemented with 10 mM EDTA and 2 mM DTT. 75 nM bacterially expressed PREPL or immunopurified PREPL was pre-incubated with 10 μM inhibitor or DMSO (0.05% final concentration) as a control at 37°C for 15 min. This was followed by a reaction with 1.5 μM FP-biotin (Santa Cruz Biotechnology, sc-215056) at 37°C for 15 min. The reaction was stopped with the addition of SDS-PAGE loading buffer and denaturing the sample at 95°C for 10 min. Western blot analysis using a Streptavidin-HRP antibody (Agilent) was performed to evaluate the potential inhibition of PREPL. Membranes were stripped and relabeled with the GST antibody (Santa Cruz Biotechnology; sc138) to detect the presence of PREPL. Serine hydrolase inhibitors were kindly provided by Dr. Ben Cravatt (Scripps Research Institute, USA). Palmostatin B was purchased from Merck (cat. 178501). 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile was purchased from Vitas-M Laboratory (STK649251).
Gst antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The GST antibody is a laboratory reagent used for the detection and purification of proteins fused with the glutathione S-transferase (GST) tag. The GST antibody is a specific and high-affinity binding reagent that recognizes the GST tag, allowing for the identification and isolation of GST-tagged proteins from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fp biotin, by Santa Cruz Biotechnology (1 mentions)
Palmostatin b, by Merck Group (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Phospho creb, by Cell Signaling Technology (1 mentions)
Luteolin, by Merck Group (1 mentions)
Cnbr sepharose 4b, by Merck Group (1 mentions)
Recombinant histone h3, by Roche (1 mentions)
Histone h3 phospho ser10 antibody, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Avantor (1 mentions)
C digit scanner, by LI COR (1 mentions)
Gfp antibody, by Santa Cruz Biotechnology (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Acetylated lysine antibody, by Santa Cruz Biotechnology (1 mentions)
Pxr antibody, by Santa Cruz Biotechnology (1 mentions)
6 protocols using gst antibody
1
Inhibition Assay for PREPL Serine Hydrolase
2
Phosphorylation-specific Antibodies for Musashi Proteins
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Antisera for phosphorylation specific human Msi2 S278 and human Musashi2 S303 were generated by immunizing rabbits with either the peptide CGFPGANS(phospho)PGPVADL or CVGNYISAAS(phospho)PQPGSGF (Proteintech Group Inc.). The antibodies were purified over a peptide-affinity column and used for western blot analyses at 1:1000 dilution. Abcam antibodies to Msi1 were used at 1:1000. Sigma Tubulin antibodies were used at 1:20,000 and Cell Signaling GAPDH antibodies were used at 1:10,000. The phospho-specific MAP kinase antibody (Cell Signaling) was used at 1:1000 to detect activating phosphorylation at Thr202 and Tyr204. The antibody to detect total MAP kinase (Cell Signaling) was used at 1:1000. The GST antibody (Santa Cruz) was used at 1:1000.
3
Luteolin, Eupatilin, and Wogonin Kinase Assay
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Luteolin was of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). Eupatilin and Wogonin were provided from Dr. Baek's Lab in Kyung-Hee University. These compounds were prepared in DMSO (Sigma-Aldrich). [32P-γ] ATP was purchased from Perkin Elmer/NEN (Waltham, MA, USA) and recombinant histone H3 was purchased from Roche Applied Science (Indianapolis, IN, USA). Other recombinant proteins such as glutathione sulfotransferase (GST), GST-VRK1, GST-aurora kinase B (AURKB) and His-BAF were expressed in E.coli (BL21) and were purified by affinity chromatography. Lamin B, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and GST antibody was purchased from Santa Cruz Technology (Santa Cruz, CA, USA) and histone H3 phospho-Ser10 antibody was purchased from Abcam (Cambridge, UK) and those against histone H3, phospho-CREB and CREB were obtained from Cell Signaling Technology (Danvers, MA, USA). VRK1 and phospho-BAF (gift from Robert Craigie, NIH, USA) antibody were generated in rabbit using purified VRK1 proteins. Hoechst 33342 was purchased from Sigma-Aldrich. CNBr-Sepharose 4B was purchased from Sigma-Aldrich.
4
In vitro Nuclear Export Assay
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We performed in vitro nuclear export experiment with slight modifications (Cassany and Gerace, 2009 (link)). First, 1 µM GST-NESPKI-NLSIBB (GST-NES-NLS), 1 µM KPNB1, 1 µM NTF2, 1 × energy regeneration system (Cassany and Gerace, 2009 (link)), 0.01% Triton-X100, and 2 µM of hRan were added to semi-permeabilized HeLa cells and incubated at room temperature for 60 mins to accumulate nuclear cargoes. The very low concentration of Triton-X prevents non-specific cytoplasmic binding, but does not permeate nuclear envelopes (Figure 3—figure supplement 1 ). After washing, the cells were incubated with either human or yeast proteins (0.1 µM CRM1, 1 µM Ran and 1 µM RanBP1), energy regeneration system, 0.01% Triton-X, for different time points at room temperature with gentle shaking. After reaction, the cells were washed, fixed and visualized by immunostaining with GST antibody (Santa Cruz, 1:400). Statistics were based on measurements from at least 30 cells for each sample, and statistical significance was calculated by one-way ANOVA test in Graphpad software.
5
Western Blot Analysis of Transfected Proteins
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For Western blotting, transfected cells were harvested and lysed in lysis buffer (50 mM Tris-Cl (pH-8.0), 150 mM NaCl, 1 mM DTT, 5 mM EDTA, 0.5% NP-40 and 1X protease inhibitor cocktail (Amresco) followed by sonication. Protein samples were resolved by SDS-PAGE, transferred onto the methanol-charged PVDF membrane using semi-dry transfer apparatus. Subsequently, the blots were probed with indicated primary antibodies. RFP antibody, GFP antibody, GST antibody, PXR antibody, His antibody and acetylated-lysine antibody were purchased from Santa Cruz Biotechnologies. TIP60 antibody, RXRα antibody and β-actin antibody were procured from Cell Signaling Technology (CST). Acetylated Histone H4 and Histone antibodies were purchased from Millipore. Horseradish peroxidase-conjugated (HRP) anti-rabbit and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnologies while light chain specific HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Millipore. Blots were developed using ECL reagent (BioRad) and C digit scanner (Licor).
6
Protein–Protein Interaction Assay
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