Cells were lysed with base lysis buffer containing 1% IGEPAL CA-630, 200mM NaCl, and 50mM Tris pH 8.0 on ice for 30 minutes. The base lysis buffer was supplemented with the protease inhibitors aprotinin (5ug/mL), leupeptin (5ug/mL), sodium fluoride (0.9mM), dithiothreitol (DTT, 1mM), sodium vanadate (1mM), and beta-glycerophosphate (20mM). Lysates were centrifuged for 15 minutes at 4°C to recover supernatant and quantified for protein concentration using Protein Assay Dye Reagent Concentrate. 40ug of protein was loaded per well for polyacrylamide gel electrophoresis using Mini-PROTEAN Precast Polyacrylamide Gels (Bio-Rad). Western blotting was performed using low fluorescence PVDF membrane (Bio-Rad). Transfer was accomplished using 1X Towbin Transfer Buffer Containing 20% methanol at 300mA for 1 hour. Blots were blocked for 1 hour using Intercept (TBS) Blocking Buffer (LI-COR Biosciences), before incubation with primary antibody overnight at 4°C. Blots were incubated for 1hr at room temperature with IRDye Secondary antibodies and were visualized by near infrared fluorescence via Li-COR Odyssey CLx imager. The antibodies used for westerns were: ß-actin (1:1000), phospho-S6 (Ser235/236) (1:2000), S6 (1:1000), phospho-ACC (1:1000), ACC (1:1000), phospho-AMPK (1:1000), AMPK (1:1000), phospho-ULK1 (Ser777) (1:700), HIF-1α (1:1000), and phospho-STAT3 (1:2000).
Low fluorescence pvdf membrane
Manufactured by Bio-Rad
Sourced in United States
The low fluorescence PVDF membrane is a specialized product designed for applications where low background fluorescence is required. It is a type of membrane made of polyvinylidene fluoride (PVDF) material that exhibits reduced fluorescence properties, making it suitable for various analytical techniques that rely on fluorescence detection.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Image lab 6, by Bio-Rad (3 mentions)
Image studio lite quantification software, by LI COR (2 mentions)
Rabbit flag tag ab, by Merck Group (2 mentions)
Irdye 680rd goat anti mouse igg, by LI COR (2 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Criterion stain free 4 20 gels, by Bio-Rad (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Pvdf membrane, by Beyotime (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab97051, by Abcam (2 mentions)
23 protocols using low fluorescence pvdf membrane
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of SOCS3, JAK1, and JAK2 Proteins
3
Evaluating EGFR, STAT3, AKT, and ERK Signaling
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Anti-EGFR antibody, anti-STAT3 antibody, anti-AKT antibody, and anti-ERK1/2 antibody were purchased from Abcam. Fetal calf serum (FCS) was obtained from Gibco (USA). Phospho-STAT3, phospho-AKT, and phospho-ERK1/2 antibodies were purchased from CST company (USA). Bovine Serum Albumin (BSA) and PVDF membranes were purchased from Beyotime Biotechnology (Shanghai, China). Cell culture plates were purchased from Corning (New York, USA). DMEM, hypoxanthine-aminopterin-thymidine (HAT), and HT were purchased from Invitrogen (California, USA). The low-fluorescence PVDF membrane was purchased from Bio-Rad Laboratories. Unless otherwise specified, reagents were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA). Animal Experiments were performed under a project license (No.: 20200506) granted by animal ethics committee of First Hospital of Shanxi Medical University, in compliance with national guidelines for the care and use of animals.
4
SDS-PAGE and Western Blot Analysis
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Cell monolayers were lysed in 2× Laemmli sample buffer and boiled for 5 min prior to SDS-PAGE using Mini-PROTEAN TGX Stain-Free precast gels (Bio-Rad). After gel activation, as per the manufacturer's instructions, proteins were transferred to a low fluorescence PVDF membrane (Bio-Rad), blocked and incubated with primary and secondary antibodies (given above). Membranes were then incubated for 5 min in the Clarity enhanced chemiluminescence western blotting substrate (Bio-Rad) and imaged on a Bio-Rad ChemiDoc. The integrated intensity of immunolabeled protein bands was calculated using Bio-Rad Image Lab software and normalized by Bio-Rad stain-free UV tryptophan labeling.
5
Western Blot Analysis of Histone Acetylation
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Example 5
Proteins were separated on Criterion Stain-Free 4-20% gels (Biorad 567-8095) at 200V for 50 min. Proteins were transferred to low fluorescence PVDF membrane (Biorad 162-0264) at 0.14 amps for 60 min. Gels and membranes were imaged with a Chemidoc XRS system (Biorad 170-8265) for quality control purposes. Membranes were processed as follows: blocked in Tris buffered saline+Tween 20 (TBST, 0.1% Tween 20) containing 5% blocker (Biorad 170-6404) overnight at 4° C. The following steps were performed at room temperature: The membrane was washed in TBST, incubated with primary antibodies in TBST containing 1% blocker (acetyl histone H3 lysine 9: EMD Millipore 06-942-S 1:4000, acetyl histone H4 lysine 12: EMD Millipore 07-595 1:4000) for 60 min, washed in TBST, incubated with secondary antibody in TBST containing 1% blocker (anti-rabbit-HRP: Cell Signaling #7074S 1:5000, anti-mouse-HRP: Cell Signaling #7076S 1:5000) for 60 min, washed in TBST, developed with ECL prime western blotting detection reagent (GE RPN2232), and visualized with a Chemidoc XRS system. Western blot images were converted from Image Lab 5.2.1 (.scn) files to 600 dpi .tif files. The images were opened in ImageJ. Images were converted to 8-bit and background was subtracted with a rolling ball radius of 50.0 pixels. Images were inverted and mean band intensity was quantified with the measurement tool.
6
Western Blot Analysis of HDAC Proteins
7
Western Blot Analysis of SOCS3, JAK1, and JAK2 Proteins
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Precipitated protein samples were centrifuged at maximum speed for 25 min at 4°C. Protein pellets were dried at 60°C for 10 min and resuspended in 1× SDS-PAGE Laemmli sample buffer containing 2.5% 2-ME. Proteins were resolved in a 12% Laemmli gel and transferred to a low-fluorescence PVDF membrane (Bio-Rad). The presence of WT or mutant SOCS3–Strep, DDK-JAK1, and DDK-JAK2 proteins in the elution fractions was detected in the same PVDF membrane using mouse Strep-Tag-II mAb (Novagen) and rabbit FLAG tag Ab (Sigma-Aldrich) (1:2000 dilution) as primary Abs and IRDye 680RD goat anti-mouse IgG (LI-COR Biosciences) and IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences) (1:12,000 dilution) as secondary Abs. Image analysis was performed using the Image Studio Lite Quantification Software (LI-COR Biosciences).
8
Western Blot Analysis of BMAL1 Protein
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Total cell protein was isolated from cells using lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.2% SDS, supplemented with protease inhibitors). Equal protein quantities were run on an SDS 4 to 20% TGX miniprotean TGX stain-free protein gel (Bio-Rad) and transferred to low fluorescence PVDF membrane (Bio-Rad) via the Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with Odyssey blocking buffer (Li-Cor) for 1 h at room temperature. Samples were probed with antibody overnight at 4 °C (BMAL1-D2L7G, Cell Signaling Technologies 14020). Immunoreactivity was visualized using the Odyssey CLx near-infrared imaging system and fluorescence quantified in ImageStudioLite (Li-Cor).
9
Western Blot Analysis of Immunoprecipitated Samples
10
Cytoplasmic and Nuclear Protein Analysis
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