Total protein was extracted from cells and tissues using RIPA buffer (Beyotime, China). Then, the protein concentration was detected by a BCA assay kit (Santa Cruz, USA). Protein samples (40 μg) were separated by SDS‒PAGE and then transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibodies overnight at 4 °C and with a goat anti-mouse IgG H&L (HRP)-preadsorbed secondary antibody for 1 h at room temperature. Finally, enhanced chemiluminescence (ECL, Thermo Fisher, MA, USA) was used to visualize the membrane. Protein band analysis was conducted with ImageJ software. The following antibodies were used: anti-Granzyme B (1:1000, ab283315, Abcam, USA); anti-METTL14 (1:1000, ab220030, Abcam, USA); anti-HSD17B6 (1:1000, orb539880, Biorbyt, USA); anti-Perforin (1:1000, ab47225, Abcam, USA); anti-GAPDH (1:2000, ab8245, Abcam, USA); and goat anti-mouse IgG H&L (HRP)-preadsorbed secondary antibody (1:5000, ab47827, Abcam, USA).
Bca assay kit
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The BCA assay kit is a colorimetric detection method for determining the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) compound to produce a purple-colored reaction that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Ab47827, by Abcam (1 mentions)
Ab220030, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Mouse anti human gapdh monoclonal antibody sc 137179, by Santa Cruz Biotechnology (1 mentions)
Mouse anti human zeb1 monoclonal antibody sc 81428, by Santa Cruz Biotechnology (1 mentions)
Mmp 9, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Anti cleaved caspase 9, by Abcam (1 mentions)
Anti ddx5, by Abcam (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti cox 2, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti bax, by Abcam (1 mentions)
6 protocols using bca assay kit
1
Western Blot Analysis of Protein Expression
2
Western Blot for ZEB1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ice-cold radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was adopted to isolate total protein from tissues or cells. The total protein concentration was examined using a BCA assay kit (Santa Cruz Biotechnology). Equal quantities of total protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA), and then blocked in TBS/0.1% Tween (TBST) containing 5% milk at room temperature for 2 h. The membranes were incubated at 4°C overnight with primary antibodies: mouse anti-human ZEB1 monoclonal antibody (sc-81428; 1:1,000 dilution; Santa Cruz Biotechnology) or mouse anti-human GAPDH monoclonal antibody (sc-137179; 1:1,000 dilution; Santa Cruz Biotechnology). After being washed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2005; 1:5,000 dilution; Santa Cruz Biotechnology) at room temperature for 2 h. Finally, an enhanced chemiluminescence reagent (Bio-Rad Laboratories, Hercules, CA, USA) was used to detect protein signals. GAPDH was used as internal control.
3
Western Blot Analysis of Apoptosis and Extracellular Matrix Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were rifted in RIPA lysis buffer plus PMSF with low temperature, and BCA assay kit (Santa Cruz, California, USA) examined total protein concentration. Prepared protein samples were separated in SDS-PAGE and transferred into 0.22 μm PVDF membranes and then were incubated with prepared antibodies. Finally, this membrane was visualized by enhanced chemiluminescence (ThermoFisher, MA, USA). The antibody against β-actin was purchased from CST (Beverly, Massachusetts, USA). Antibodies against Bax, Bcl-2, MMP-2, and MMP-9 were purchased from Abcam (MA, USA).
4
Protein Expression Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted with ice-cold RIPA lysis buffer plus PMSF. Total protein concentrations were detected with BCA assay kit (Santa Cruz, California, USA). Prepared protein samples were separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred into 0.22 μm PVDF membranes. After blocked with skim milk, these membranes were incubated with primary antibodies at 4 °C overnights, followed by incubation with the appropriate secondary antibody for 1 h at room temperature. Finally, the enhanced chemiluminescence (ECL, Pierce, Rockford, IL) visualized this membrane. The primary antibodies were anti-PCNA, anti-Ki-67, anti-Bcl-2, anti-Bax, anti-cleaved caspase-3, anti-cleaved caspase-9, anti-Cox-2, anti-MMP-2, anti-MMP-9, anti-DDX5 and anti-β-actin (Abcam, Cambridge, UK).
5
Examining miR-140-5p Regulation in HepG2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 cells were seeded in 24-well plates and were transfected with NC mimics and miR-140-5p mimics for 48 h. Cells were then cracked in RIPA lysis buffer plus PMSF in low temperature. BCA assay kit (Santa Cruz, California, USA) were used to detect total protein concentration. Prepared protein samples were separated in SDS-PAGE, transferred into 0.22 μm PVDF membranes and incubated with ready antibodies. The primary antibody was incubated overnight, and the secondary antibody for 2 h. Finally, enhanced chemiluminescence (Thermo Fisher, MA, USA) visualized this membrane. The antibody β-actin was purchased from CST (1:1000 dilution, Beverly, Massachusetts, USA). Antibodies against the GSY1, PPP1CC were purchased from Abcam (1:1000 dilution, Shanghai, China).
6
Molecular Profiling of Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AGS and SGC-7901 cell lines transfected with NC mimic and miR-498 mimic were collected and then cracked in RIPA lysis buffer plus PMSF in low temperature. After extracted, the total protein concentration was detected by BCA assay kit (Santa Cruz, USA). Prepared protein samples were loaded in SDS-PAGE and then transferred into 0.22μm PVDF membranes and incubated with prepared antibodies, the primary antibody incubated overnight, and the secondary antibody for 2 h. Finally, enhanced chemiluminescence (Thermo Fisher Scienti c, USA) visualized this membrane. The antibody β-actin was purchased from CST (Beverly, USA). Antibodies against the Cyclin D1, CDK2, P21, Cox-2, MMP-2, MMP-9 and FOXK1 were purchased from Abcam (Shanghai, China).
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!