Cells were harvested and lysed in 1 × RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail. The protein concentrations of each extracted protein sample were measured using Bio-Rad protein assay reagents. A total of 30 micrograms of each protein sample was subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% skim milk and then incubated with specific primary antibody overnight. The antibody-probed membrane was then washed 6 times with 1 × PBST (0.05% Tween 20 in 1 × PBS). After washing, the appropriate horseradish peroxidase-labeled secondary antibodies were added to the membrane for 1 h, and then washed with 1 × PBST. The bound antibodies were detected by enhanced chemiluminescence (ECL) reagents (GE Healthcare Life Sciences; Uppsala, Sweden). The blot signals were visualized by X-ray film (Roche Applied Science, Mannheim, Germany). The intensities of signals were quantified by GeneTools software (SYNGEN, Cambridge, UK).
X ray film
Manufactured by Roche
Sourced in Germany
X-ray film is a photographic film used to capture and record images during X-ray examinations. It is designed to be sensitive to the energy emitted by X-ray radiation, allowing the creation of detailed images of the internal structures of the body.
Automatically generated - may contain errors
Lab products found in correlation
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Positively charged nylon membrane, by Roche (2 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Sypro ruby protein gel stain, by Thermo Fisher Scientific (1 mentions)
Typhoon 9400 scanner, by Cytiva (1 mentions)
Anti lamin b, by Santa Cruz Biotechnology (1 mentions)
Gs 800 densitometer, by Bio-Rad (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti bip, by Santa Cruz Biotechnology (1 mentions)
Anti pcaf, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti ha, by Merck Group (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Nuclear protein and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
β actin mouse monoclonal antibody, by Beyotime (1 mentions)
Rbp j, by Santa Cruz Biotechnology (1 mentions)
12 protocols using x ray film
1
Protein Extraction and Western Blot
2
Western Blot Analysis of PSCA Protein
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Cultured PC3 cells were trypsinized and dissolved in 1 M lysis buffer (2.4 gr urea, 5.1 gr thiourea, 4 gr CHAPS, 4 ml of Tris buffer in 10 ml deionized water) and protease inhibitor cocktail (Sigma, MO, USA). Protein concentration in the supernatant was assayed by the BCA kit. Cell lysates and/or rPSCA protein (50 μg) were blended and boiled by loading buffer for 5 min and loaded onto 7% SDS–PAGE plus 10% SDS and β-mercaptoethanol. The proteins were transferred onto the polyvinylidene difluoride (PVDF) membrane (240 mA for 45 min at 4°C) in transfer buffer of a wet western blot apparatus; the membrane was then blocked for 1.5 h at RT (21 ). Afterward, 1:1000 dilution of anti-PSCA antibody (0.8 mg/ml) was added to the membrane in TBS, incubated overnight at 4°C and washed six times with TBS-T buffer. The goat anti-rabbit HRP-conjugated antibody (1:60000, Abcam, MA, USA) was then added for 1 h at RT. Finally, bands were exposed by enhanced chemiluminescence (ECL) (Pierce) and presented on an x-ray film (Roche).
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Cultured PC3 cells were trypsinized and dissolved in 1 M lysis buffer (2.4 gr urea, 5.1 gr thiourea, 4 gr CHAPS, 4 ml of Tris buffer in 10 ml deionized water) and protease inhibitor cocktail (Sigma, MO, USA). Protein concentration in the supernatant was assayed by the BCA kit. Cell lysates and/or rPSCA protein (50 μg) were blended and boiled by loading buffer for 5 min and loaded onto 7% SDS–PAGE plus 10% SDS and β-mercaptoethanol. The proteins were transferred onto the polyvinylidene difluoride (PVDF) membrane (240 mA for 45 min at 4°C) in transfer buffer of a wet western blot apparatus; the membrane was then blocked for 1.5 h at RT (21 ). Afterward, 1:1000 dilution of anti-PSCA antibody (0.8 mg/ml) was added to the membrane in TBS, incubated overnight at 4°C and washed six times with TBS-T buffer. The goat anti-rabbit HRP-conjugated antibody (1:60000, Abcam, MA, USA) was then added for 1 h at RT. Finally, bands were exposed by enhanced chemiluminescence (ECL) (Pierce) and presented on an x-ray film (Roche).
3
Protein Separation and Detection by SDS-PAGE
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Purified proteins were separated by SDS–PAGE (NuPAGE gel 4–12% Bis-Tris, Invitrogen) and visualized either by Coomassie staining or SYPRO Ruby Protein Gel Stain (Invitrogen) using a Typhoon 9400 scanner (Amersham Biosciences) with λex = 457 nm and λem = 610 nm. Alternatively, proteins were separated by SDS–PAGE on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and blotted onto polyvinylidene difluoride (PVDF) membranes (Pierce). Proteins were revealed using the following antibodies at the indicated dilutions: i) polyclonal anti-MPK10 antibody, generated by rabbit immunization using recombinant MPK10 protein produced in E. coli transformed with pGEX-Strep3-MPK10 plasmid (Eurogentec), 1∶10,000; ii) anti-phospho-tyrosine antibody 4G10 Platinum from Millipore, 1∶1,000; and iii) secondary goat anti-rabbit-HRP and anti-mouse-HRP antibodies from Thermo Scientific, 1∶20,000. The visualization was performed on X-ray film (Roche) at various exposure times.
4
Co-Immunoprecipitation of EBV LMP1 Complexes
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293T cells were transiently co-transfected with indicated expression plasmids and the cell lysates were prepared two days post-transfection for Co-IP. To get the high levels of ectopic expression, 293T, a highly transfectable derivative of HEK293, was chosen for the Co-IP study. The IP kit was purchased from Roche and Co-IP was performed according to the manufacturers' instructions. The immunoprecipitated complexes were analyzed by Western blotting. Western blotting was carried out according to the standard protocols. The horseradish peroxidase-conjugated secondary antibodies were purchased from Pierce. After antibody incubation, the blots were washed and incubated with SuperSignal West Pico Substrate (Pierce), and the chemiluminescent signal was detected using an x-ray film (Roche). The film was then scanned, and the protein bands were quantified by the GS-800 densitometer (Bio-Rad). The antibodies used in this study include: anti-EBV LMP1 (ETU001, KeraFAST), anti-XBP-1 (sc-7160), anti-BiP (sc-1501), anti-PCAF (sc-8999), anti-lamin B (sc-373918, Santa Cruz Biotechnology), anti-acetylated lysine (ab21623), anti-GCN5 (ab71965, Abcam), anti-HA (H3663, Sigma), anti-actin (MAB1501, Millipore), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118S, Cell Signaling).
5
Crude Total Protein Extraction and Western Blot Analysis
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For crude total protein extraction, cells were lysed in 1X buffer (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) for 10 min and centrifuged at 12,000 ×g for 10 min at 4°C to obtain the soluble proteins. Twenty-five to fifty micrograms of protein were separated on a 12% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% nonfat milk, incubated with various primary antibodies overnight, and then washed with 1X PBST solution (0.05% Tween 20 in 1X PBS). After washing, the appropriate secondary antibodies, which were each labeled with horseradish peroxidase, were added to the membrane for 1 h and then washed with 1X PBST solution. The antigen-antibody complexes were detected using Amersham™ ECL™ Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences; Uppsala, Sweden). Autoradiographic signals were detected by an X-ray film (Roche Applied Science, Mannheim, Germany). The signal intensity was quantitated by GeneTools analysis software (SYNGEN, Cambridge, UK).
6
Western Blot Analysis of LV Proteins
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Left ventricular tissues were ground in liquid nitrogen. Nuclear protein and cytoplasmic protein extracts were made using a Nuclear Protein and Cytoplasmic Protein Extraction kit (Beyotime Biotechnology, Beijing, China) according to the manufacturer's instructions. The protein concentrations were determined with a BCA Protein Assay Kit (Beyotime Biotechnology, Beijing, China). Samples containing equal amounts of protein (20 µg) were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking for 1 h at room temperature, the membranes were probed with rabbit antibodies against RBP-J (1:500), bax (1:500), bcl-2 (1:500) (Santa Cruz Biotechnology, CA, USA), cleaved-caspase 3 (1:1000) (Abcam, Cambridge, UK), and β-actin mouse monoclonal antibody (1:1000) (Beyotime Biotechnology, Beijing, China) overnight at 4°C, followed by incubation with secondary horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:1000) (Beyotime Biotechnology, Beijing, China) or horseradish peroxidase-conjugated anti-mouse IgG antibody (1:1000) (Beyotime Biotechnology, Beijing, China) for 1 h at room temperature. Proteins were detected by exposing the blots to X-ray film (Roche Applied Science).
7
Protein Expression Analysis of mESCs
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Following treatment with or without Icaritin, mESCs were washed three times with ice-old PBS. Total protein was extracted by cell lysis in Lammeli’s lysis buffer containing 1% Triton X-100, and scraped by a cell lifter. Protein concentration from the cell lysate was examined by BCA protein assay (Pierce). 30 μg protein was denatured, resolved in 12% SDS-PAGE minigel, and electrophoretically transferred onto an Immobilon-P membrane (Millipore). The membrane was pre-blocked with 5% nonfat dry milk for 1 h and hybridized with antibodies against OCT4, NANOG, SOX2, KLF4, p130, CDX2, Cyclin E and CDK2 (all from Cell Signaling), at 4 °C overnight with agitation. The membrane was washed extensively with 0.1% Tween-20 in PBS, and then incubated with horse-radish peroxidase conjugated secondary antibodies (Molecular Probes) in 1:8000 dilution at room temperature for 1 h. Protein expression signals were detected with enhanced chemiluminescence (GE Healthcare) and developed on an X-ray film (Roche).
8
Western Blot Analysis of Protein Samples
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Cells were lysed in an ice-cold buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 2 mM EDTA, 1% NP-40, 10% glycerol, 1 mM DTT, 1x protease inhibitor cocktail and 1x phosphatase inhibitor cocktail at 4°C for 30 min. Cell lysates were separated on a sodium dodecylsulfate (SDS)-polyacrylamide gel, and then transferred electrophoretically onto the Hybond-C Extra nitrocellulose membrane (GE Healthcare, Piscataway, NJ, USA). The membrane was pre-hybridized in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween-20 (TBST buffer), and 5% skim milk for 1 h, and then transferred to a solution containing 1% bovine serum albumin (BSA)/TBST and a primary antibody and incubated overnight at 4°C. After washing with the TBST buffer, the membrane was submerged in 1% BSA/TBST containing a horseradish peroxidase-conjugated secondary antibody for 1 h. The membrane was washed with TBST buffer, and then developed with an enhanced chemiluminescence (ECL) system (Perkin-Elmer, Boston, MA, USA) and exposed to x-ray film (Roche, Indianapolis, IN, USA).
9
Southern Blot Analysis of Transgene Integration
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For additional confirmation of transgene integration, Southern blot analysis was performed with two PCR positive events using the method described in [14 (link)]. Briefly, gDNA was extracted from leaf tissues using the CTAB extraction method. Twenty micrograms of gDNA were digested overnight at 37 °C with HindIII, which cuts only once within the T-DNA region of the plasmid vectors used in transformation. Digested DNA was separated by electrophoresis in 0.8% agarose gel. The DNA was blotted onto a positively charged nylon membrane (Roche Diagnostics, Lewes, East Sussex, UK) by upward capillary in a 20× SSC buffer. A digoxigenin (DIG)-dUTP labelled nptII probe was hybridized to the blotted DNA. Hybridizing bands corresponding to the nptII gene were visualized by exposure to an X-ray film (Roche Diagnostics, Lewes, East Sussex, UK) for 30 min.
10
Western Blot Analysis of Protein Expression
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Cells were scraped and lysed in a lysis buffer. The lysis buffer is PRO-PREP protein extraction solution. It is a commercial solution and purchased from iNtRon Biotechnology (Cat No. 17081.1, iNtRon Biotechnology, Gyeonggi, Korea). The lysates were centrifuged at 12,000× g at 4 • C for 10 min. The supernatants were acquired. Exactly 50 µg of proteins was subjected to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk, soaked overnight with several primary antibodies and cleaned with 1 × PBS with Tween ® 20 (PBST) solution (Sigma-Aldrich Corp., Saint Louis, MO, USA). After cleaning, the appropriate secondary antibodies conjugated to horseradish peroxidase were added to the polyvinylidene fluoride membrane, incubated for 1 h and then cleaned with 1 × PBST solution. The dilution for the first antibodies was 5% bovine serum albumin. The dilution for the secondary antibodies was PBST solution. The antigen-antibody complexes were evaluated by Amersham TM ECLTM prime Western blotting detection reagent (GE Healthcare Life Sciences; Uppsala, Sweden). Autoradiographic signs were revealed by X-ray film (Roche, Mannheim, Germany). The sign intensity was quantified by GeneTools analysis software (SYNGEN, Cambridge, UK).
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