The concentration of total proteins was measured with a BCA protein assay (Thermo Fisher Scientific Inc., Waltham, MA, USA) and equal amounts of samples (100 mg protein) were separated by electrophoresis using 15% polyacrylamide gels and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Pittsburgh, PA, USA) following the manufacturer's instructions. The membranes were incubated with mouse-derived anti-GAPDH antibodies (1:500 in Tris-Buffered Saline with Tween-20 (TBST), Beyotime Institute of Biotechnology, Shanghai, China) and mouse-derived anti-His antibodies (1:3000 in TBST, Sigma, Santa Clara, CA, USA) for 1.5 h at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (1:5000 in TBST, Beyotime Institute of Biotechnology) at room temperature for 1 h. Reaction with chemiluminescence substrate luminal reagent (GE Healthcare) and exposure to X-ray film were used to examine the immunolabeled bands. The optical density of the bands was scanned and quantified with ImageJ software version 1.40g (http://rsb.info.nih.gov/ij/ , NIH), and histogram analysis using the Origin 9.5 software (http://www.originlab.com/ ).
Tris buffered saline with tween 20 tbst
Manufactured by Beyotime
Sourced in China
Tris-Buffered Saline with Tween-20 (TBST) is a buffer solution commonly used in various biochemical and molecular biology applications. It consists of a Tris buffer, sodium chloride, and the non-ionic detergent Tween-20. TBST is primarily used as a wash buffer to maintain the stability and activity of proteins during laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Tbs t, by Merck Group (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence substrate luminal reagent, by GE Healthcare (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Na k atpase, by Beyotime (1 mentions)
Gapdh, by Beyotime (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by PerkinElmer (1 mentions)
Bca kit, by Beyotime (1 mentions)
Horseradish peroxidase linked secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ripa lysis solution, by Beyotime (1 mentions)
Antibody diluent, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Vazyme (1 mentions)
4 protocols using tris buffered saline with tween 20 tbst
1
Western Blot Protein Analysis Protocol
2
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ARPE-19 cells cultured on scaffolds for total protein extraction were washed with cold PBS before being lysed with RIPA lysis buffer at 4°C for 30 minutes. Cell lysates were centrifuged, and the supernatant was collected. The protein concentrations were determined by the BCA protein assay kit (Beyotime, China). The same amount of proteins in each sample was mixed with a loading buffer and heated at 100°C for 5 min to denature proteins. The protein samples were dissolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After treatment with 5% w/v blotting grade blocker, the membranes were incubated with corresponding primary antibodies: Na+/K+-ATPase and GAPDH (diluted at 1:1000 v/v in Tris Buffered Saline with Tween® 20 (TBST), Beyotime, China). The corresponding signals were then captured using horseradish peroxidase (conjugated secondary anti-rabbit IgG antibodies, 1:5000 dilution v/v, Beyotime, China) on a chemiluminescence imaging system. Quantification of bands was performed using ImageJ software, and data were analyzed and compared. Western blot was performed in triplicates in three independent experiments.
3
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed using a 2X loading lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The total amount of proteins from the cultured cells was subjected to 12% SDS-PAGE and transferred onto a hybrid polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Following inhibition with 5% (w/v) non-fat dried milk in Tris-buffered saline with Tween-20 (TBST; Beyotime Institute of Biotechnology), the PVDF membranes were washed four times (15 min each) with TBST at room temperature and incubated with primary antibodies, including rabbit anti-human Ki67 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-human BACE1, Aβ1–40, Aβ1–42 and GAPDH antibodies (Cell Signaling Technology, Inc., Beverly, MA, USA). Following extensive washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig) G secondary antibody (1:1,000; Santa Cruz Biotechnology, Inc.) for 1 h. Following washing four times (15 min each) with TBST at room temperature, the immunoreactivity was visualized using an enhanced chemiluminescence kit from Perkin Elmer, Inc. (Norwalk, CT, USA).
4
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole proteins from cell lysates and lung tissues were extracted using RIPA lysis solution (P0013B, Beyotime, Shanghai, China) according to the manufacturer’s instructions and measured using a BCA kit (P0009, Beyotime, Shanghai, China). Equal amounts of protein were separated on 8%–12% SDS–PAGE gels (Vazyme, Nanjing, China), blotted onto PVDF membranes (Merck Millipore, Germany), blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 (TBST) (Beyotime, Shanghai, China) for 1 h, and incubated overnight at 4°C in antibody diluent (Beyotime, Shanghai, China) containing the following primary antibodies: rabbit antibody against p21 (1:1,000 dilution), rabbit antibody against p16 (1:1,000 dilution), rabbit antibody against p-p38MAPK (Thr180/Tyr182) (1:1,000 dilution), rabbit antibody against p38MAPK polyclonal antibody (1:1,000 dilution); mouse antibody against p53 (1:5,000 dilution); mouse antibody against GAPDH (60004-1-Ig, 1:50,000 dilution; ProteinTech); and mouse antibody against ß-Tubulin (10094-1-AP, 1:2,000 dilution; ProteinTech). The membranes were incubated with horseradish peroxidase-linked secondary antibody (Jackson Labs, United States) for 1 h, and protein expression was detected using ECL (Vazyme, Nanjing, China). Images were captured using the Tanon 5,200 imaging system (Shanghai, China). ImageJ software (version 2.0.0) was used for gray value analysis.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!