The AML12 cells were seeded at a concentration of 3×106 cells/ml into 60 mm dishes and, once confluent, were lysed in ice-cold lysis buffer containing 1% NP-40, 50 mM Tris-HCl, 0.1% SDS, 1% sodium deoxycholate and 150 mM NaCl (pH 7.4; Tianjin Chemical Reagent Factory, Tianjin, China) and centrifuged at 12,000 × g at 4°C for 3 min. The protein content was determined using Quick Start™ Bradford kit (5000202EDU; Bio-Rad Laboratories, Inc., Hercules, CA, USA), using bovine serum albumin (BSA) as the standard. Subsequently, 15 µl of the proteins were separated by SDS-PAGE on 12% acrylamide gels, following which proteins were transferred onto a PVDF membrane (EMD Millipore, Bethesda, MA, USA). The membrane was then incubated overnight in a blocking buffer containing appropriate diluent (ab64211; Abcam, Cambridge, UK) of primary antibodies against gp78 (ab54787; 1:1,000, Abcam), cidec (ab77115; 1:1,000, Abcam), PPAR-γ (ab41928; 1:1,000, Abcam) and β-actin (ab8226; 1:1,000, Abcam) at 4°C. The proteins were detected by incubation with horseradish peroxidase-conjugated secondary antibody (ab6789; 1:2,000, Abcam) in diluent (ab64211; Abcam, Cambridge, UK) at room temperature for 1 h. Following extensive washing with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, the bands were visualized using enhanced chemiluminescence and autoradiography.
Quick start bradford kit
Manufactured by Bio-Rad
Sourced in United States
The Quick Start Bradford kit is a colorimetric assay for determining the concentration of protein in solution. It utilizes the Bradford method, which is based on the color change of Coomassie Brilliant Blue G-250 dye in response to different concentrations of protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab64211, by Abcam (1 mentions)
Ab54787, by Abcam (1 mentions)
Ab77115, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab41928, by Abcam (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Amylose resin, by New England Biolabs (1 mentions)
6 protocols using quick start bradford kit
1
Protein Expression Analysis in AML12 Cells
2
Quantification of SDF-1 Protein in Kidney Tissues
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Kidney tissues were powdered in liquid nitrogen and lysed with radioimmunoprecipitation assay (RIPA) buffer (Cyto Matin Gene, Isfahan, Iran) according to the manufacturer’s instructions. Protein concentrations were measured using Quick Start Bradford kit (BioRad, Hercules, California, USA) with bovine serum albumin, employed as standard. A concentration of 5 μg/mL of all samples was prepared for enzyme- linked immunosorbent assay (ELISA) assay. SDF-1 protein was quantified with RayBio® SDF-1 α ELISA kit (RayBiotech, Norcross, Georgia, USA) and Epoch™ Microplate Spectrophotometer (BioTek, Winooski, Vermont, USA).
3
WSMV Protein-RNA Binding Assay
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Maltose binding protein (MBP)-tagged proteins of WSMV P1 or P1-W303A were expressed in E. coli through pMAL-c5X and purified by affinity chromatography with amylose resin (New England Biolabs, Ipswich, MA, USA). Affinity purified proteins were quantified by the Bradford assay using Quick Start Bradford kit (Bio-Rad). PTGS-like ds-siRNAs were prepared from chemically synthesized 21- or 24-nt 5’P-ssRNA with 2-nt 3’ overhangs (Integrated DNA Technologies, Skokie IL) from GFP sequence. These ds-siRNAs were diluted to 1 µM concentration in 20 mM Tris-HCl (pH 8.0), 1 mM EDTA and 50 mM NaCl (TEN buffer). Long dsRNA was prepared by annealing in vitro transcribed positive- and negative-sense ssRNAs derived from GFP (180-nt) or WSMV CP (600-nt) sequences in TEN-T buffer (TEN buffer containing 0.02% Tween 20).
Synthetic ds-siRNAs or ss/dsRNA (100 ng) was incubated with MBP-tagged protein (0.3 nmol) in 30 µL of TEN buffer for 30 min at room temperature. Following incubation, the reaction mixture was analyzed through 4–12% non-denaturing polyacrylamide gel electrophoresis at 4 °C and visualized with SYBR Green stain (Thermo Scientific, Waltham, MA, USA) as described in Samuel et al. [47 (link)]. ImageJ software was used to quantify the fluorescent signal of unbound dsRNA bands [48 (link)].
Synthetic ds-siRNAs or ss/dsRNA (100 ng) was incubated with MBP-tagged protein (0.3 nmol) in 30 µL of TEN buffer for 30 min at room temperature. Following incubation, the reaction mixture was analyzed through 4–12% non-denaturing polyacrylamide gel electrophoresis at 4 °C and visualized with SYBR Green stain (Thermo Scientific, Waltham, MA, USA) as described in Samuel et al. [47 (link)]. ImageJ software was used to quantify the fluorescent signal of unbound dsRNA bands [48 (link)].
4
Purification of His-tagged Proteins
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After induction, the cells were harvested by centrifugation (4°C, 8000 × g, 5 min), resuspended in ice‐cold binding buffer (pH 8.0, 20 mM Tris–HCl, 500 mM NaCl) and lysed by ultrasound (10 min, 2 s off, 2 s on). Supernatants were prepared through centrifugation (4°C, 8000 × g, 15 min). The supernatants were purified using Ni2+ His‐tag column with non‐linear imidazole gradient from 20 to 300 mM (in resuspension buffer). The active fractions were pooled and dialyzed for 24 h in deionized water at 4°C. Then, the purified enzyme was lyophilized and stored at −20°C. Ultimately, the lyophilized enzyme was dissolved in a buffer containing 20 mM Tris–HCl (pH 8.0) as the protein solution for further experiments. The purity of targeted proteins was estimated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis. Protein concentrations were determined using Quick Start™ Bradford Kit (Bio‐Rad, USA).
5
Quantitative Analysis of cADPR in HEK293 Cells
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HEK293 cells carrying the inducible SARM1 expression cassette were transiently transfected with the plasmids encoding the YFP-fusion nanobody, C6-YFP or 1053-YFP, by PEI. Forty-eight hours post-transfection, the cells were lysed with 0.6 M perchloric acid. After centrifugation, the pellets were re-dissolved in 1 M NaOH and quantified by Bradford assay (Quick StartTM Bradford Kit, BIO-RAD), and the supernatants containing cADPR were neutralized with Chloroform/Tri-n-octylamine mixture (volume ratio 3:1) and treated with NADase (home-made). The concentration of cADPR was analyzed by the cycling assay as described previously37 (link). The results were presented as pmol cADPR/mg proteins. The protein expression level was quantified by western blot with Anti-GFP (TranGen HT801-01, 1:1000).
6
SARM1 Complex Structure Analysis
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HEK293 cells carrying the inducible SARM1 expression cassette were transiently transfected with the plasmids encoding the YFP-fusion nanobody, C6-YFP or 1053-YFP, by PEI. Forty-eight hours post-transfection, the cells were lysed with 0.6 M perchloric acid. After centrifugation, the pellets were re-dissolved in 1 M NaOH and quantified by Bradford assay (Quick Start TM Bradford Kit, BIO-RAD), and the supernatants containing cADPR were neutralized with Chloroform/Tri-n-octylamine mixture (volume ratio 3:1) and treated with NADase (home-made). The concentration of cADPR was analyzed by the cycling assay as described previously 35 (link) . The results (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
were selected and subtracted with mask that left only "one layer" of the "two-layer" SARM1-C6 complex, leading to a single-layer hexadecimer composed of Nb-C6 and SARM1. The final construction of these subtracted particles was carried out with C8 symmetry, resulting in a final map of >5Å resolution around most part of ARM and TIR domain. Local resolution was estimated by Resmap, map sharpening was performed with lowest resolution for auto-B fit set as 3.5Å.
were selected and subtracted with mask that left only "one layer" of the "two-layer" SARM1-C6 complex, leading to a single-layer hexadecimer composed of Nb-C6 and SARM1. The final construction of these subtracted particles was carried out with C8 symmetry, resulting in a final map of >5Å resolution around most part of ARM and TIR domain. Local resolution was estimated by Resmap, map sharpening was performed with lowest resolution for auto-B fit set as 3.5Å.
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