The proteins were obtained with RIPA Lysis (Sangon, China) and separated using 12% SDS-PAGE gel electrophoresis at 100 V for 1 h. After equilibrating the protein-containing gel with the transfer buffer, the protein was transferred to the PVDF membrane (Sangon, China). The hybridization membrane was then incubated with anti-TTK (1:1000, Sangon, China) and β-actin (1:1000, Sangon, China) for 2 h and blocked at 4°C overnight. After 24-hincubation, the membrane was treated with a secondary antibody (Rabbit-Mouse; Sangon, China) for 2 hours. ImageJ software was eventually used to quantify the intensity of bands.
Ripa lysis
Manufactured by Sangon
Sourced in China
RIPA lysis buffer is a strong detergent-based lysis buffer used for the extraction and solubilization of proteins from cell and tissue samples. It contains a combination of ionic and non-ionic detergents that effectively lyse a variety of cell types, allowing the release and extraction of proteins for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Sangon (2 mentions)
β actin, by Sangon (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Af5145, by Affinity Biosciences (1 mentions)
Af0127, by Affinity Biosciences (1 mentions)
Cy5997, by Abways (1 mentions)
Af0136, by Affinity Biosciences (1 mentions)
Cy5621, by Abways (1 mentions)
Cy6617, by Abways (1 mentions)
S0009, by Affinity Biosciences (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
Bca assay, by Beyotime (1 mentions)
Nitrocellulose membrane, by Sangon (1 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Akt antibody, by Thermo Fisher Scientific (1 mentions)
P akt, by Thermo Fisher Scientific (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Bca kit, by Sangon (1 mentions)
P pi3k, by Affinity Biosciences (1 mentions)
4 protocols using ripa lysis
1
Western Blot Analysis of TTK Protein
2
Quantitative Analysis of Extracellular Matrix Proteins
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Total proteins were extracted from colon tissue or kidney tissue samples when treated with radioimmunoprecipitation assay (RIPA) lysis (Sangon Biotech, Shanghai, China). The concentrations of total proteins were measured by bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). The proteins were separated on SDS–polyacrylamide gel electrophoresis and transferred to an NC membrane. The NC membrane was blocked with 5% non-fat milk and then incubated with primary antibodies against ZO-1 (AF5145, 1:500, Affinity), occludin (CY5997, Abways), claudin-1 (AF0127, 1:1,000, Affinity), Col-I (AF0127, 1:2,000, Affinity), Col-III (AF0136, 1:1,000, Affinity), FN (CY5621, 1:1,000, Abways), and LN (CY6617, 1:1,000, Abways) overnight at 4°C, respectively. Then, the membrane was washed with TBST and incubated with secondary goat anti-rabbit (S0001, 1:5,000, Affinity) and goat anti-rat antibodies (S0009, 1:5,000, Affinity) at 37°C for 2 h (Cell Signaling Technology, CA, USA). Finally, the proteins were monitored with enhanced chemiluminescence reagents (GE Healthcare Life Sciences, NJ, USA) and then quantitatively analyzed by ImageJ software (version 1.4.0., National Institutes of Health). GAPDH protein was used for the internal control.
3
Protein Extraction and Quantification
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RIPA lysis (Sangon) was applied at 48 h after incubation to extract total protein. BCA kit (Sangon) was applied to measure the concentration of total protein. The solution containing 20 μg protein was subjected to 4-20% SDS-PAGE gel (Willget, Shanghai, P. R. China), and followed by transferring to a nitrocellulose membrane (Sangon). The membrane was blocked by 5% skim milk at 37℃ for 1 h and subsequently cultivated with primary antibodies at 4℃ overnight. Then, the membrane was incubated with HRP-linked secondary antibody (Cell Signaling Technology, Boston, MA, USA) for 1 h at 37℃. Lastly, the membrane was visualized by ECL kit (Beyotime, Shanghai, P. R. China). The following primary antibodies were used: TRPC1 antibody (1:2,000, Invitrogen), PI3K antibody (1:1,000, Affinity, Changzhou, P. R. China), p-PI3K (1:1,000, Affinity), AKT antibody (1:2,500, Invitrogen), p-AKT (1:2,500, Invitrogen) and β-actin (1:1,500, Invitrogen). The Image J v 1.8 (NIH, Bethesda, MD, USA) was applied to quantify protein bands. Protein expression was the ratio of target protein grey density against control protein density, with 3 bands of each protein being analyzed.
4
Protein Extraction and Western Blotting
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Total proteins were extracted by RIPA lysis (Cat#: C500005, Sangon, China). Proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred to PVDF membranes (Cat#: F019532, Sangon, China). After blocking the membranes in 5% skim milk, the hybridization membrane was incubated with primary antibodies RBBP4 (Cat#: D154089, Sangon, China) and GADPH (Cat#: D190090, Sangon, China) at 4 °C overnight. The membranes were continued to incubate with the corresponding secondary antibody for 2 hours, and the protein was detected using ECL chemiluminescence kit (Cat#: C510043, Sangon, China) according to the reagent instructions.
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