As described in our previous report (Wang et al., 2014 (link)), protein was extracted and separated on 12% SDS-PAGE (Beyotime Biotechnology, Shanghai, China), and then transferred onto polyvinylidene fluoride membranes (Merck Millipore, Temecula, CA, United States) by electroblotting. The membranes were blocked with 5% BSA for 2 h at room temperature and incubated with the following primary antibodies overnight at 4°C: prdx1 (1:1000, Bosterbio, Pleasanton, CA, United States), Bcl2 (1:1000, Abcam), Bax (1:1000, Abcam), and β-actin (1:1000, Santa Cruz Biotechnology, Dallas, TX, United States). The membranes were washed with TBS-T washing buffer and incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:10000, Zsbio, Beijing, China) or anti-mouse secondary antibodies (1:10000, Zsbio) at 25°C for 2 h. Bound antibodies were visualized using an enhanced chemiluminescence (ECL) substrate and gray values were evaluated with ImageJ software.
Anti mouse secondary antibody
Manufactured by ZSGB-BIO
Sourced in China
Anti-mouse secondary antibodies are laboratory reagents used to detect the presence of primary antibodies that have been raised against mouse antigens. They provide a universal detection system for mouse-derived primary antibodies in various immunoassays and applications.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Sds page, by Beyotime (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by ZSGB-BIO (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Akr1b1, by Boster Bio (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
β tubulin, by Abmart (1 mentions)
Protein lysis buffer, by Beyotime (1 mentions)
Zb 2301, by ZSGB-BIO (1 mentions)
Zb 2305, by ZSGB-BIO (1 mentions)
Anti rabbit secondary antibody, by ZSGB-BIO (1 mentions)
Biotinylated secondary antibody, by ZSGB-BIO (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Enhanced chemiluminescence ecl assay kit, by Cytiva (1 mentions)
Fitc conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti mouse secondary antibody
1
Western Blot Protocol for Protein Analysis
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
Whole-cell protein was extracted using a protein lysis buffer(Beyotime, Shanghai, China).25μg samples were loaded in the well and were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Then blocked with 5% nonfat milk powder in TBST (TBS with 0.1% Tween) for 2 hours at room temperature and incubated with the appropriate primary antibody at 4°C overnight. Antibodies used include AKR1B1(1:50; BOSTER, Wuhan, China) and β-tubulin(1:4000; Abmart, Shanghai, China). The next day these membranes were washed with TBST (TBS with 0.1% Tween) three times for 5 minutes each. Then membranes were incubated with HRP-coupled anti-rabbit secondary antibodies (1:1000; ZSGB-BIO, Beijing, China) and anti-mouse secondary antibodies (1:1000; ZSGB-BIO, Beijing, China) at room temperature for 2 hours. Next, membranes were washed with TBST (TBS with 0.1% Tween) three times for 5 minutes each. Finally use ECL reagents (Beyotime, Shanghai, China) and visualize by an enhanced chemiluminescence detection system (Thermo Scientific, Waltham, MA, USA).
3
Western Blot Analysis of ANGPTL4 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The main procedure was performed as described in our previous report [41 (link)]. In brief, the total cell protein lysates were obtained by RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, pH 7.4) containing protease inhibitors. A total of 20 μg of protein lysates from each lane were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Then, the proteins were transferred to PVDF membranes. After blocking with 5% nonfat dry milk, the membranes were incubated with primary antibodies. ANGPTL4 (A2011, 1:1000) and GAPDH antibodies (TA-08, 1:1000) were purchased from ABclonal and ZSGB-BIO, respectively. The anti-rabbit secondary antibody (ZB-2301, 1:5000) and anti-mouse secondary antibody (ZB-2305, 1:5000) were purchased from ZSGB-BIO.
4
Osteopontin Regulates HEC-1A Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human uterine epithelial cell line HEC-1A cells were acquired from the American Type Culture Collection (ATCC). Recombinant OPN protein (rhOPN) was purchased from R&D Systems. Rabbit anti-AKT and phosphor-AKT (Ser473) antibodies were purchased from Bioworld Technology. Rabbit anti-ERK, phospho-ERK 1/2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and U0126 (a specific inhibitor of MEK/ERK; 10-5M) and LY294002 (a specific inhibitor of p38; 10-6M) were purchased from Beyotime Institute of Biotechnology. MMP-2, E-cadherin, N-cadherin and Vimentin were purchased by Proteintech. Cell Counting Kit-8 was purchased by Dojindo. Enhanced chemiluminescence (ECL) assay kit was purchased from Amersham. Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody, anti-mouse secondary antibody, biotinylated secondary antibody, streptavidin-horseradish peroxidase and diaminobenzidine (DAB)-peroxidase substrate were purchased from ZSGB-BIO. TRITC-conjugated goat anti-rabbit secondary antibody IgG and FITC-conjugated goat anti-mouse secondary antibody were purchased from Thermo.
Cell lines and culture conditions HEC-1A cells were grown in McCoy's 5A supplement with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were maintained at 37 o C under 5% CO 2 in humidified air.
Cell lines and culture conditions HEC-1A cells were grown in McCoy's 5A supplement with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were maintained at 37 o C under 5% CO 2 in humidified air.
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!