For protein extract preparation, cells were lysed on ice with RIPA Lysis Buffer (ThermoFisher) containing complete protease and phosphatase inhibitor cocktail (Roche). Soluble protein extracts were separated by centrifugation at 13000 rpm for 15 min and diluted in Laemlli sample buffer. The obtained cell lysates were resolved on sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and transferred on polyvinylidene difluoride membrane Hybond TM-P (Amersham Bioscience). Membranes were saturated with 5% bovine serum albumin at room temperature for 2 h and incubated with the following primary antibodies at 4 °C overnight. Primary antibodies were used as follows: ELF3 (1:1000, AF5787; R&D Systems, Minneapolis, USA), EHF (1:1000, 27195-1-AP; Proteintech, Illinois, USA), TGIF1 (1:1000, ab52955; Abcam, Masseachusettes, USA), β-actin (1:1000, 3700S; CST, Boston, USA) and GAPDH (1:1000, 2118S; CST, Boston, USA). Secondary anti-mouse IgG (ab175775), anti-rabbit IgG (ab175773), and anti-goat IgG (ab175776) all conjugated to Alexa Fluor 680 (Abcam, Masseachusettes, USA) were incubated with the membranes for 2 h at room temperature at 1:10,000 dilution. All bands of western blot were detected and qualified with gray scale ratio by Odyssey CLx imaging systems (LI-COR, Nebraska, USA).
Hybond p
Manufactured by Cytiva
Sourced in United States, United Kingdom, Sweden, Germany
Hybond-P is a polyvinylidene difluoride (PVDF) membrane used in Western blotting applications. It is designed for the transfer and immobilization of proteins from polyacrylamide gels to a solid support for subsequent detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (5 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (4 mentions)
Ecl plus kit, by Cytiva (4 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Bca assay, by Thermo Fisher Scientific (3 mentions)
Protein assay kit, by Bio-Rad (3 mentions)
Enhanced chemiluminescence detection kit, by Cytiva (3 mentions)
Sc 1350, by Santa Cruz Biotechnology (3 mentions)
Sc 59993, by Santa Cruz Biotechnology (3 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Ecl detection system, by Cytiva (3 mentions)
Fusion fx7 imaging system, by Avantor (3 mentions)
Amersham imager 600 system, by GE Healthcare (3 mentions)
Seeblue plus2, by Thermo Fisher Scientific (3 mentions)
Protein assay, by Bio-Rad (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Anti ve cadherin goat polyclonal antibody, by Santa Cruz Biotechnology (2 mentions)
Anti claudin 5, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
118 protocols using hybond p
1
Protein Extraction and Western Blotting
2
GFP-NanoLuc Detection in P. falciparum
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For Western blot analysis of GFP-NanoLuc expression, total protein extracts from in vitro-cultured mixed asexual blood stage P. falciparum parasites (line Exp245 clone1) were separated via 12% (w/v) SDS-PAGE gel and transferred to a PVDF transfer membrane (Amersham Hybondtm-P) by electroblotting. GFP-NanoLuc expression was detected by incubating the membrane with rabbit polyclonal anti-GFP antibody (1:1000; Abcam; ab290), followed by incubation with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:10,000; GE Healthcare; NA934V). Immunostained proteins were visualised by incubating the membrane with Pierce ECL Plus Substrate (Thermo Scientific; 32132). The resulting chemiluminescent signals were captured using X-ray film (SuperRX-N Fuji Medical).
3
Tight Junction Protein Analysis
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Cultured cells were harvested by lysis in RIPA buffer for detection of the tight junction proteins. Lysates were centrifuged at 15,000×g for 15 min at 4 °C, supernatants were collected, and protein concentrations were determined with the BCA protein assay reagent (Pierce, Rockford, IL, USA). An equal amount of protein for each sample was separated on a 4–15% TGX (Tris–Glycine eXtended) gel (Bio-Rad, USA), transferred onto HybondTM-P (Amersham, UK), and incubated with antibodies. Anti-claudin-5, anti-occludin, and anti-ZO-1 mouse monoclonal antibodies (Invitrogen, USA) were used at dilutions of 1:5,000. Anti-VE-cadherin goat polyclonal antibody (Santa Cruz, USA) was used at a dilution of 1:2,500. Anti-β-actin mouse monoclonal antibody (Sigma, additional loading control) was used at a dilution of 1:10,000 in 3% bovine serum albumin in PBS. To visualize the immunoreactive bands, blots were incubated in SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology) and were detected using a FluorChem SP Imaging System (Alpha Innotech Corp., USA).
4
Western Blot Analysis of DAP12 Protein
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Cell lysates were extracted with lysis buffer (1% Triton-X 100, protease inhibitor cocktail (CompleteTM, Roche Diagnostics, Indianapolis, IN, USA), 700 ng/ml pepstatin A (Peptide Institute, Osaka, Japan) in phosphate-buffered saline). Aliquots were electrophoresed on 10% polyacrylamide sodium dodecyl sulfate-gel and blotted onto polyvinylidene difluoride membrane (HybondTM-P, Amersham Biosciences, Piscataway, NJ, USA). The membranes were probed with anti-DAP12 antibodies (Santa Cruz (Dallas, TX, USA)), followed by HRP-conjugated secondary antibodies IgG (Cell Signalling technology, Danvers, MA, USA). Immunoreactive bands were visualized with ImmunoStar LD (Fuji Film) and detected on a LAS-4000 mini (Fuji Film).
5
Tight Junction Protein Detection
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Cultured cells were harvested by lysis in RIPA buffer for detection of the tight junction proteins. Lysates were centrifuged at 15,000 × g for 15 min at 4°C, supernatants were collected, and protein concentrations were determined with the BCA protein assay reagent (Pierce, Rockford, IL, USA). An equal amount of protein for each sample was separated on a 4-15% TGX (Tris-Glycine eXtended) gel (Bio-Rad, USA), transferred onto HybondTM-P (Amersham, UK), and incubated with antibodies. Anti-claudin-5, antioccludin, and anti-ZO-1 mouse monoclonal antibodies (Invitrogen, USA) were used at dilutions of 1:5,000. Anti-VE-cadherin goat polyclonal antibody (Santa Cruz, USA) was used at a dilution of 1:2,500. Anti-βactin mouse monoclonal antibody (Sigma, additional loading control) was used at a dilution of 1:10,000 in 3% bovine serum albumin in PBS. To visualize the immunoreactive bands, blots were incubated in SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology) and were detected using a FluorChem SP Imaging System (Alpha Innotech Corp., USA).
6
Quantification of OPG and RANKL Proteins
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In order to confirm gene expression results, a representative sample (eight gingival biopsies) was analyzed for OPG and RANKL proteins quantification, by mechanical and sonication process, quantified by Bradford methods (19) . Five-hundred µg of protein were homogenate in sample buffer (0.02 mM dithioreitol; 1.38 mM sodium dodecyl sulfate; 125 mM Tris-HCl, [pH 6.8 and 20% glycerol]) and 50 µg of protein were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were then electro-blotted to a PVDF membrane (Hy-bond TM-P, Amersham Biosciences, Brazil). Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline 0.001% Tween 20 for 1 h and then incubated with the primary antibodies: rabbit-polyclonal anti-OPG (Abcam, Cambridge, UK), rabbit-polyclonal anti-RANKL (Abcam) and mouse-monoclonal anti-tubulin (Clone DM1A; Sigma-Aldrich, St. Louis, MO, USA) overnight. The reaction was observed using SuperSignalWest Pico Chemiluminescent Substrate (Pierce).
7
Quantifying Osteogenic Markers in ADSC
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Western blot analysis was used to detect OC and type I collagen. ADSCs were collected 7 or 14 days after culture from stable transfections of plasmid pEGFP-N1-BMP-2 (group A), or without transfection (group C). The protein lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Hybond TM -P; Amersham Biosciences, Munich, Germany). Membranes were blocked with 0.2% Tween 20 and 5% non-fat dry milk in PBS. The lysates were then incubated with primary antibodies (1:200) for 2 h at room temperature followed by 3 washes with PBS. A horseradish peroxidase-labeled secondary antibody was then added and incubated for 1 h at room temperature followed by 3 washes with PBS and detected by X-ray film. The results were repeated three times and b-actin was used as internal control.
8
Protein Expression Analysis Protocol
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Whole cell extracts or nuclear extracts were obtained as reported in Mazzarelli et al. 2007. Acid soluble proteins were extracted according to the protocol for histone proteins (UpState). Total and acid proteins extracted as above (10μg) were equally loaded (Ponceau S-staining) on 10% SDS- Proteins were transferred to a PVDF membrane (Hybond P, Amersham GE Healthcare). Primary antibodies: anti-CPT1A (Santa Cruz, H95), anti-haemoagglutinine mouse monoclonal antibody (Covance), anti-acetyl-histone H4 (Upstate) rabbit polyclonal antibodies, anti-HDAC1 (UpState, clone 2E10) mouse monoclonal antibodies, anti-HDAC2 /HDAC3 /HDAC7 (Cell Signaling Thecnology, Antibody Sampler Kit) rabbit polyclonal antibodies, anti-SirT1 (Cell Signaling Thecnology, C14H4) rabbit monoclonal antibodies and anti-Cleaved Caspase9 (Cell Signaling Thecnology, Asp330) rabbit polyclonal antibody were used. Filters were reprobed with anti-β-actin mouse monoclonal antibodies (Sigma-Aldrich, 63103 USA) or anti-Sp1 mouse monoclonal antibodies (Santa Cruz biotechnologies, Inc) to normalize respectively cytoplasmic or nuclear protein levels. Filters were developed using an enhanced chemiluminescence system (ECL, Amersham-GE Healthcare).
9
Western Blot Analysis of Jak2/Stat3 Pathway
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Cells were homogenized in cell lysis buffer. Samples were separated by SDS-PAGE. Separated proteins were transferred onto nitrocellulose membranes (Hybond-P, Amersham Biosciences; Piscataway, NJ, USA) that were incubated overnight at 4°C using the following antibodies: JAK2 (dilution 1:1,000, Cell signal, Danvers, MA, USA), p-JAK2 (dilution 1:1,000, Cell signal), STAT3 (dilution 1:1,000, Cell signal), p-STAT3 (1:200,000, Abcam), Pim-1 (dilution 1:10,000, Abcam). Membranes were then washed and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Blot bands were visualized with an enhanced chemiluminescene system (Amersham Bioscience, Fairfield, Connecticut, USA). The protein bands were analyzed using VisionWorks LS, version 6.7.1.
10
Western Blot Analysis of MxA Protein
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Whole cell lysates were boiled for 10 min in 4× NuPAGE LDS loading buffer (Invitrogen) and cooled on ice. Samples were loaded onto precast 4–12% NuPAGE Bis‐Tris gels and transferred onto PVDF membranes (Hybond‐P, Amersham Biosciences). Blocked membranes were probed using rabbit anti‐MxA (Abnova) or mouse anti‐β‐actin (Sigma) antibodies. For fluorescent detection the blocking step and all antibody incubations were performed in LI‐COR blocking buffer. Membranes were allowed to dry and proteins were detected using the Odyssey Western Blot Scanner (LI‐COR).
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