Western blot analysis was performed to measure protein expression. Briefly, radio-immunoprecipitation assay (RIPA) lysis buffer was used to collect total protein from HUVECs and mesenteric arteries. One 6-cm-dish of cells were lysed with 150 μl of RIPA lysis buffer, and 0.1 grams of mesenteric arterial tissue were lysed with 100 μl of RIPA lysis buffer, lysed on ice for 45 min, and then centrifuged at 12,000 g at 4°C for 25 min. The supernatants were collected, and protein concentrations were determined. Next, proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto transfer membranes (Millipore). After blocking with 5% nonfat milk for 60 min at room temperature, the membranes were incubated with targeted primary antibodies overnight at 4°C. After washing in tris-buffered saline with Tween-20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After washing the membranes three times in TBST, protein bands were detected using enhanced chemiluminescence reagents (Bio-Rad). Densitometric analysis was conducted using Bio-Rad software.
Transfer membranes
Manufactured by Merck Group
Sourced in United States
Transfer membranes are porous materials used in various laboratory techniques, such as Western blotting, Northern blotting, and Southern blotting. They are designed to transfer biomolecules, such as proteins or nucleic acids, from a gel-based separation medium to a solid support for further analysis or detection.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
A600669, by Sangon (2 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Anti chop, by Abcam (1 mentions)
Anti ciap1, by Abcam (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using transfer membranes
1
Protein Expression Analysis by Western Blot
2
Western Blot Protein Analysis in Kidney and Cells
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Total proteins were obtained from the kidney and cells using protein extraction reagent containing protease and phosphatase inhibitor cocktail (Pierce Chemical Co., Rockford, IL, USA). After quantification of the protein concentration by Bradford method, proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto transfer membranes (Millipore, Billerica, MA, USA), as previously described [23] (link). The primary antibodies were anti-TRIM13 (1:1000, Abcam, Cambridge, UK), anti-CHOP (1:1000; Abcam), anti-cIAP1 (1:1000; Abcam), and anti-β-actin (1:1000; Cell Signaling Technology, Danvers, MA, USA). After incubation with the horseradish peroxidase-labeled secondary antibody (1:3000; Abcam) for 1 h, an enhanced chemiluminescence kit (Pierce Chemical, Rockford, IL, USA) was used to visualize the protein bands on the transfer membranes.
3
Western Blot Protein Analysis Protocol
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Cells were washed with PBS, lysed in lysis buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol) and boiled for 3min. The protein concentrations of the lysates were quantified with a BCA Protein Assay Kit (Thermo Fisher). Protein samples were separated by SDS-PAGE using 10% agarose gel and transferred to transfer membranes (Millipore-Sigma, St. Louis, MO, USA), which were incubated in 5% nonfat milk (A600669, Sangon) at room temperature for 2 hours. The membrane was incubated with the primary antibody at 4°C overnight or at room temperature for 2 hours. The membrane was then incubated with horseradish-peroxidase-conjugated secondary antibody or Alexa-647-conjugated secondary antibody at 25°C for 1 hour as described previously [68 (link)]. Anti-LANA antibody was purchased from Millipore-Sigma (St. Louis, MO, USA) and an Anti-K-Rta antibody was described previously [69 (link)].
4
Western Blot Analysis of Protein Samples
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Cells were washed with PBS, lysed in lysis buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol) and boiled for 3min. The protein concentrations of the lysates were quantified with a BCA Protein Assay Kit (Thermo Fisher). Protein samples were separated by SDS-PAGE using 10% agarose gel and transferred to transfer membranes (Millipore-Sigma, St. Louis, MO, USA), which were incubated in 5% nonfat milk (A600669, Sangon) at room temperature for 2 hours. The membrane was incubated with the primary antibody at 4°C overnight or at room temperature for 2 hours. The membrane was then incubated with horseradish-peroxidase-conjugated secondary antibody or Alexa-647-conjugated secondary antibody at 25°C for 1 hour. For cell fractionation, cells were suspended with hypotonic buffer (20mM Tris-HCl(pH 7.4), 10mM NaCl, 3mM MgCl2, 0.5mM DTT and proteinase inhibitor cocktail) for 15min on ice and 0.5% NP-40 was added. Cells were then centrifuged for 10 minutes at 3000rpm to collect the supernatants (cytoplasm fraction). Pellets were washed with PBS 2 times and suspended with protein lysis buffer (nuclear fraction).
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