Protein samples were harvested from flash frozen chicken brainstem tissue. Samples were homogenized in EDTA buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% Glycerol, 5% β-ME, 10 mM EDTA) using the Ultra-Turrax® T10 homogenizer (IKA® Works, Inc., Wilmington, NC). 50 μg of protein lysate in SDS buffer (2% SDS, 50 mM Tris pH 7.6, 5% glycerol, and 0.025% bromophenol blue) was incubated at 70°C for 10 minutes, resolved in NuPAGE 4–12% Bis-Tris Gels (Life Technologies, Carlsbad, CA), and then transferred onto PDVF membranes (GE Healthcare, Chicago, IL). After blocking in 5% milk in PBS with 0.05% Tween (PBS-T) for 30 minutes at room temperature, membranes were probed against the primary antibodies (Tab. 1 ) overnight at 4°C in 1% milk in PBS-T. Specific secondary HRP-conjugated antibodies were used at 1:2500 dilution (Santa Cruz, Biotechonology®, Inc., Dallas, TX) and blots were developed with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, Inc., Waltham, MA) and exposed to X-ray film.
Pdvf membrane
Manufactured by GE Healthcare
Sourced in United States
PVDF (Polyvinylidene Fluoride) membranes are a type of laboratory equipment used for various filtration and separation processes. These membranes are made from a thermoplastic polymer, known for their chemical resistance, thermal stability, and mechanical strength. They are commonly used in applications such as microfiltration, ultrafiltration, and protein purification, among others.
Automatically generated - may contain errors
Lab products found in correlation
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (4 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Ultra turrax t10 homogenizer, by IKA Group (3 mentions)
Bca protein colorimetric assay kit, by Elabscience (1 mentions)
Protease inhibitor cocktail, by Solarbio (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Np 40 lysis buffer, by Solarbio (1 mentions)
Ultraturrax t10, by IKA Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Complete edta free protease inhibitor cocktail, by Merck Group (1 mentions)
Rabbit anti histone h3, by Abcam (1 mentions)
Rat anti aid, by Active Motif (1 mentions)
Af596, by R&D Systems (1 mentions)
Alexa fluor 488 donkey anti goat secondary antibody, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
P0160, by Agilent Technologies (1 mentions)
A9418, by Merck Group (1 mentions)
Enhanced chemiluminiscence reagent, by GE Healthcare (1 mentions)
12 protocols using pdvf membrane
1
Western Blot Analysis of Chicken Brainstem Proteins
2
Western Blot Analysis of Protein Expression
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NP-40 lysis buffer (Solarbio, China), PMSF(Solarbio, China) ,and Protease Inhibitor Cocktail(Solarbio, China)were used to extract proteins. Total proteins were transferred to PDVF membranes (GE, America) from 10% SDS–PAGE after determining the protein content (BCA Protein Colorimetric Assay Kit, Elabscience, China). Membranes were incubated in primary antibodies α-SMA (1:1000), Smad2 (1: 2000), Smad3 (1: 1000), phospho-Smad2 (1: 5000), phospho-Smad3 (1: 2000), and p38MAPK (1: 500) at 4 °C overnight. After washing, membranes were incubated with secondary antibodies (Rabbit anti-Goat IgG–HRP, abisin, China at room temperature for 2 h. Chemiluminescence was performed with Thermo ECL (elabscience, China and visualization was done with a Tanon gel imager. We used the Image J software to quantify using β-actin as an internal standard. This experiment was repeated three times to obtain average values.
3
Western Blot Analysis of FMRP in Mouse Brain
4
Protein Extraction and Western Blot Analysis
5
Chicken Brainstem Protein Extraction and Analysis
6
Western Blot Methodology for Protein Analysis
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The Western blot procedures were performed as we described earlier [28 (link),37 (link),38 (link),43 (link)]. Briefly, cells were lysed for 30 min on ice in a radioimmunoprecipitation assay (RIPA) buffer (Sigma Aldrich, St. Louis, MO, USA, #R0278) supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Sigma Aldrich, #11873580001). Proteins were analyzed by 4%–12% Bis-Tris NuPAGE gels (Invitrogen, Carlsbad, CA, USA, #NP0322), transferred to PDVF membranes (GE Healthcare, Boston, MA, USA, #GE10600023), and probed with indicated antibodies. Rat anti-AID (1:500, Active Motif, Carlsbad, CA, USA, #39886); mouse anti-GCN5, clone A-11 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, #sc-365321); rabbit anti-PCAF, clone C14G9 (1:1000, Cell Signaling Technology, Leiden, The Netherlands, #3378); rabbit antihistone H3 (1:1000, Abcam, Cambridge, UK, #ab1791); rabbit antihistone H3 acetyl K9 (1:500, Abcam, Cambridge, UK, #ab32129).
7
Adenoviral Transduction of HEK293 Cells
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HEK293 cells were infected with a multiplicity of infection (MOI) of 2 or 5 for 24–48 h with Ad5.CMV.sIL21R and Ad5.CMV.Null or Ad5.CMV.eGFP as negative controls. For immunocytochemistry, anti-IL21R antibody (AF596, R&D Systems, Minneapolis, MN, USA) and Alexa Fluor 488 donkey anti-goat secondary antibody (A11055, Invitrogen, Carlsbad, CA, USA) were used at a dilution of 1:100 and 1:200, respectively. For immunodetection, cell pellet was homogenized in lysis buffer (RIPA) and total protein was determined from lysed cells and supernatants using the Pierce BCA Protein Assay (232,227, Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts and supernatants were loaded onto denaturing acrylamide gels and then electrotransferred to PDVF membranes (10,600,023, GE Healthcare, Chicago, IL, USA). Anti-IL21R primary antibody (AF596, R&D Systems) was used at 1:1000 dilution and goat HRP-anti-IgG secondary antibody (P0160, Dako, Agilent Technologies, Santa Clara, CA, USA) at 1:10,000 were used in the presence of 5% (w/v) of BSA (A9418, Sigma-Aldrich, St. Louis, MO, USA).
8
Western Blot Protein Detection
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Protein extracts (15-25 μg per sample) from tissue samples were run in denaturing acrylamide gels, and then electrotransferred to PDVF membranes (GE Healthcare, Barcelona, Spain). Membranes were blocked with TBS-T (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2% Tween-20) containing 5% skimmed milk, and incubated with the primary K113 antibody. Detection was performed with an appropriate horseradish peroxidase-conjugated secondary antibody (EZBiolab, Carmel, IN, USA) and enhanced chemiluminiscence reagent (GE Healthcare). The K113 antibody was used at 1/5000 dilution; polyclonal rabbit anti-actin antibody (Sigma A2066, Madrid, Spain) at 1/1000; and secondary rabbit HRP-anti-Ig antibody (DakoCytomation, P0399, Madrid, Spain) at 1/10 000. Band pixel intensities were quantified using ImageJ (Wayne Rasband National Institutes of Health, Bethesda, MD, USA) and normalised to actin levels.
9
Western Blot Protein Analysis Protocol
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Cell lysates were prepared from washed cultured cells using RIPA buffer and inhibitors (Halt protease and phosphatase Thermo scientific/Pierce, Cramlington, UK). Samples (5 μg) were separated using SDS-PAGE and 10% gels. The separated proteins were transferred onto polyvinlidene fluoride (PDVF) membrane (GE Healthcare) then washed with PBS-Tween (1xPBS with 1% Tween 20) and blocked (5% w/v non-fat dry milk). Washed membranes were incubated with primary antibody as indicated. HRP-conjugated secondary antibody (GE Healthcare) was detected using Enhanced Chemiluminescence (GE Healthcare).
10
Quantitative Analysis of VDAC Isoforms
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The total lysates from the parental and VDAC1 knock-out HAP1 cells were obtained from 106 cells in a lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, EDTA 1 mM, 1% TRITON X-100, protease inhibitors cocktail). Protein samples were separated using SDS-PAGE electrophoresis and transferred to a PDVF membrane (GE Healthcare, Chicago, IL, USA). The membranes were blocked in 5% BSA in PBS with 0.1% Tween-20 at room temperature for 1h and incubated overnight at 4 °C with the following primary antibodies: VDAC1 (1:1000, Abcam, Cambridge, UK), VDAC2 (1:200, Abcam), VDAC3 (1:100, Abcam), and β-Tubulin (1:2000, Cell Signaling, Danvers, MA, USA). After washing, membranes were incubated with IRDye-conjugated secondary antibodies (1:25,000, Li-Cor Biosciences, Lincoln, NE, USA). Signals were detected by the Odyssey CLx Imaging System (Li-Cor Biosciences) and analyzed using Image Studio Lite software (Li-Cor Biosciences). The same software was used for quantification.
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