Total proteins were extracted from HUVECs using a Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology) and the protein concentrations were quantified using a bicinchoninic acid (BCA) kit (Aspen Biotechnology Co., Ltd.). Subsequently, proteins (40 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and were then transferred onto polyvinylidene fluoride (PVDF) membranes. Following blocking with 3% skimmed milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies used were the following: Anti-Vascular endothelial cadherin (VE-cadherin; dilution, 1:1,000; cat. no. ab33168), anti-CD31 (dilution, 1:1,000; cat. no. ab222783), anti-α-smooth muscle actin (α-SMA; dilution, 1:1,000; cat. no. ab150301), anti-IGF1R (dilution, 1:1,000; cat. no. ab182408), anti-β-catenin (dilution, 1:1,000; cat. no. ab32572) and anti-β-actin (dilution, 1:1,000; cat. no. ab8226; all from Abcam). Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (dilution, 1:5,000; cat. no. ab205718; Abcam) for 1 h at room temperature (25 (link)). Finally, the blots were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Inc.). β-actin served as the internal control.
Bca kit
Manufactured by Aspen
Sourced in China
The BCA kit is a colorimetric assay used to determine the total protein concentration in a sample. It is based on the reduction of copper (II) to copper (I) by proteins in an alkaline environment, which then reacts with bicinchoninic acid to produce a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Cyclin d1, by Proteintech (2 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence, by Beyotime (1 mentions)
Anti gap43, by Abcam (1 mentions)
Anti ngf, by Abcam (1 mentions)
As1004, by Aspen (1 mentions)
Anti vacht, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Protease inhibitor, by MedChemExpress (1 mentions)
Chemidoc xrs gel imaging system, by Bio-Rad (1 mentions)
Anti l lactyl lysine, by PTM Biolabs (1 mentions)
Ripa buffer, by Aspen (1 mentions)
Radioimmunoprecipitation assay (ripa), by Aspen (1 mentions)
Alphaeasefc, by Bio-Techne (1 mentions)
Rabbit anti gapdh antibody, by Abcam (1 mentions)
Mouse anti collagen 1 antibody, by Abcam (1 mentions)
Ab267787, by Abcam (1 mentions)
Ab154598, by Abcam (1 mentions)
Ab37168, by Abcam (1 mentions)
9 protocols using bca kit
1
Protein Expression Analysis in HUVECs
2
Protein Analysis of Ischemic Myocardium
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Fifty μg of protein was extracted from the ischemic myocardial tissues at 4 weeks after MI. The tissues were lysed in radioimmunoprecipitation assay (AS1004, Aspen, Wuhan, China), and lysis buffer and protein concentrations were determined using the BCA kit (AS1086, Aspen). The proteins were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked by incubation with 5% bovine serum albumin in a TBS-Tween buffer (10 mM Tris-HCl, 150 mM NaCl, and 0.5% Tween-20) for 1 hour at room temperature, and subsequently, incubated with different primary antibodies: rabbit anti-TH (Santa, Shanghai, China), anti-GAP43 (1:1,000; Abcam, Cambridge, UK), anti-NGF (1:500; Abcam), anti-CHAT (1:500; Bioss, Beijing, China), anti-VACHT (1:500; Abcam), or anti-GAPDH (1:10,000; Abcam) overnight at 4°C. After the membrane was washed three times, the blots were incubated with secondary HRP conjugated goat anti-rabbit antibody (1:10,000; Pierce, Rockford, IL, USA) for 30 minutes at room temperature. Membranes were detected by enhanced chemiluminescence (Beyotime Biotechnology, Jiangsu, China) and exposed to film in the dark. The optical density intensity of each band was measured using AlphaEaseFC software (Alpha Innotech Corp., San Leandro, CA, USA). Results are shown as the optical density ratio to GAPDH.
3
Cell Lysate Preparation and Protein Quantification
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The procedures of cells cultivation and treatment were identical to the above mentioned. The radioimmunoprecipitation (RIPA) assay was carried out to yield the lysates of cells, which were then centrifugated at 12,000 rpm for 5 min to obtain the supernatant for immunoblot analyses. The total protein concentrations were measured by the BCA kit (Aspen Biotechnology Co., Ltd., Wuhan, China) according to the instruction provided by the manufacturer. The procedures of the electrophoresis and Western blotting analyses referenced the reported literature [33 (link)].
4
Quantifying Histone Lysine Lactylation
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Total proteins in cells were extracted using RIPA buffer (Aspen Biological, Wuhan, China) and protease inhibitors (MedChemExpress, SNJ, USA). Protein concentration was measured using the BCA kit (Aspen). Equal amounts of protein were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocked with milk at room temperature for 1 h. PVDF membranes were washed three times with TBST for 10 min each, and then incubated overnight at 4 °C with the appropriate primary antibody. Following another round of washing, the membranes were incubated with secondary antibodies at room temperature for 1 h. Finally, the membranes were developed using ChemiDoc XRS gel imaging system (Bio-Rad, Hercules, CA, USA). The antibodies used in this experiment are listed below: Anti-beta Tubulin (PTM-6414, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys18) (PTM-1427RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys14) (PTM-1414RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys9) (PTM-1419RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl Lysine (PTM-1425, PTMBIO, Zhejiang, China); Anti-Histone H3 (PTM-6621, PTMBIO, Zhejiang, China); SLC25A29 (26663-1-AP, Proteintech, Wuhan, China).
5
Western Blot Analysis of Protein Expression
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Total proteins in each sample on day 7 extracted using the protein extracting reagent RIPA (Aspen Inc.) following the manufacturer's protocol. The protein concentration was determined using the BCA kit (Aspen Inc.). Equal amounts of proteins from each sample were separated by SDS‐PAGE, transferred to PVDF membranes (Millipore Corporation, Billerica, MA) and blocked with 5% milk for 1 hr at room temperature. The membranes were incubated overnight at 4°C with rabbit anti‐GAPDH antibody (1:10,000; Abcam Inc.), rabbit anti‐VEGF‐A antibody (1:1000; Bioss Biotechnology Inc., Beijing, China), rabbit anti‐MIP‐1α antibody (1:1000; Abcam Inc.), rabbit anti‐α‐SMA antibody (1:5000) (TDY Biotechnology Inc., Beijing, China) and mouse anti‐collagen I antibody (1:500; Abcam Inc.). Finally, the membranes were subsequently incubated with the horseradish peroxidase‐conjugated goat anti‐rabbit IgG antibody (KPL) and goat antimouse IgG antibody (KPL) as secondary antibody at 1:10,000 for 30 min. The protein bands were detected by ECL chemiluminescence and quantified by AlphaEaseFC (Alpha Innotech, San Leandro, CA) software.
6
Protein Extraction and Western Blot Analysis
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Total cellular protein was extracted using lysis buffer, and protein levels were quantified using a BCA kit (ASPEN Biotechnology, Wuhan, China). Proteins were separated on a 10% SDS-PAGE before being probed with the appropriate primary antibodies for CD9, CD63 TSG101, PI3Kγ (#ab154598, Abcam, Cambridge, UK, 1:500), PIK3R1 (#4257, CST, MA, US, 1:1000), P-Akt (#4060, CST, MA, US, 1:1000), IL-17 (#sc-374218, Santa Cruz, Texas, US, 1:500), PTEN (#ab267787, Abcam, Cambridge, UK, 1:2000) and Bcl-2 (#ab182858, Abcam, Cambridge, UK, 1:1000) and then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were then probed with secondary antibodies, and protein was identified using a LiDE110 scanner (Canon, Japan). GAPDH (#ab37168, Abcam, Cambridge, UK, 1:10,000) was used for normalization, and the AlphaEaseFC software was used to measure protein band density.
7
Western Blot Analysis of Pulmonary Proteins
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Total proteins were obtained from HPASMCs and human pulmonary arteries. The concentration of proteins was assessed with a BCA kit (Aspen, Wuhan, China). Whole lysates were isolated by SDS-PAGE and transferred to PVDF membranes that were blocked by TBST with 5% non‐fat dry milk at room temperature for 1h. The membranes were first incubated with primary antibodies at 4°C overnight and then HRP-conjugated secondary antibodies at room temperature for 1h. The western ECL substrate (Bio-Rad, California, USA) and the ChemiDocTM-XRS+imaging system (Bio-Rad, California, USA) were used to visualize the bands. ImageJ software (NIH, Bethesda, MD) was used to measure the band intensities quantitatively. The following primary antibodies were used: against β-ACTIN (Sungene Biotech, Tianjin, China), GAPDH (Sungene Biotech, Tianjin, China), MBD2 (Abcam, Cambridge, UK), Cyclin D1 (Cyclin D1, Proteintech, Wuhan, Hubei, China), PCNA (Proteintech, Wuhan, Hubei, China), and BMP2 (Proteintech, Wuhan, Hubei, China).
8
Immunoblotting for MFG-E8, AKT, and Cyclin D1
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Total tissues or cell lysates were extracted from mouse lungs, pulmonary arteries, and human PASMCs; protein concentrations were determined using a BCA kit (Aspen, Wuhan, China). Whole lysates were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and were then transferred onto PVDF membranes, which were blocked with 5% BSA in Tris‐buffered saline containing 0.5% Tween‐20 for 1 hr at room temperature. The membranes were then incubated with primary antibodies overnight at 4°C before they were reacted with HRP‐conjugated secondary antibodies. An ECL reagent was used for chemiluminescent detection, and the membranes were analyzed using the ChemiDoc MP System (BioRad Laboratories, CA). Band intensities were quantitatively measured using ImageJ (NIH, Bethesda, MD). Primary antibodies against β‐actin (TDY Biotech Co., Beijing, China), mouse MFG‐E8 (R&D Systems, Minneapolis, MN), human MFG‐E8 (Abcam), p‐AKT, AKT (Cell Signaling Technology, Danvers, MA) and cyclin D1 (Proteintech Group Inc., Wuhan, China) were used.
9
Penumbra Protein Profiling for NLRP Inflammasome
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Protein samples were harvested from penumbra. Tissues were ground separately in RIPA buffer comprising protease and phosphatase inhibitors (cocktails and PMSF from Aspen) for 30 min at 4°C. A BCA kit (Aspen) was used to detect the total protein concentration of each sample. Proteins were processed by SDS-PAGE (10–12.5%) and electro-blotted onto a PVDF membrane. And the membrane was then incubated in blocking buffer (5% skim milk) for 1 h at room temperature and incubated with primary antibodies including GSDMD (Abclonal), NLRP1, NLRP3, Caspase-1 (Novus), IL-1β,IL-18 (R&D), total p-65 (Proteintech),phosphorylated p-65 (Abclonal), GAPDH (Proteintech) overnight at 4°C. After washing three times, the membrane was incubated in secondary antibody for 1 h at 24°C. The proteins were scanned with a Bio-Rad system. ImageJ software was used to quantify protein levels which were normalized to GAPDH (n = 6/group).
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