The tissue samples were homogenized in T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific, USA) and centrifuged at 10,000 g for 5 min to pellet the tissue debris. The supernatant was then collected, and the total protein was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto polyvinylidene difluoride filter (PVDF) membranes (Millipore, USA), and then blocked with 5% skim milk in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) buffer for 2 h at room temperature. Subsequently, the membranes were incubated with rabbit anti-RIOK2 polyclonal antibody (Abcam, USA; 1:100 dilution) or mouse-anti-human β-actin antibody (Abcam, USA; 1:500 dilution) as an internal control. Then, the membranes were washed in TBST and incubated with a goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibody (Abcam, USA; 1:1000 dilution) at room temperature for 2 h. Finally, the specific proteins were detected with a Novex® ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific, USA), and the membranes were exposed to films (Kodak, USA).
Mouse anti human β actin antibody
Manufactured by Abcam
Sourced in United States
The mouse anti-human β-actin antibody is a monoclonal antibody that specifically recognizes the β-actin protein, a ubiquitous and highly conserved cytoskeletal protein. This antibody can be used for the detection and quantification of β-actin in various applications, such as Western blotting, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Rabbit anti riok2 polyclonal antibody, by Abcam (1 mentions)
Novex ecl chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Rabbit anti human vegf antibody, by Abcam (1 mentions)
Ecl chemiluminescence reagent, by Beyotime (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Rabbit anti human srpk1 antibody, by Abcam (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Quercetin dihydrate, by Merck Group (1 mentions)
Peroxidase conjugated goat anti rabbit antibody, by Biosharp (1 mentions)
Rabbit anti human gapdh antibody, by Abcam (1 mentions)
Dimethyl sulfoxide (dmso), by Corning (1 mentions)
Enhanced chemiluminescence detection kit, by Merck Group (1 mentions)
P mtor, by Abmart (1 mentions)
Goat anti rabbit or anti mouse secondary antibody, by Abcam (1 mentions)
P akt, by Abmart (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
6 protocols using mouse anti human β actin antibody
1
Western Blot Analysis of RIOK2 Protein
2
Quantifying Protein Expression Levels
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1×106 cells/well were plated in a 6-well plate and grown for 24 h until about 70% confluence. After cells were treated by siRNAs for 48 h, they were harvested and lysed in RIPA buffer (Pierce, USA). Total protein was separated by polyacrylamide gel electrophoresis (PAGE) and electroblotted onto polyvinylidine difluoride filter (PVDF) membranes (Millipore, USA), followed by blocking with 5% skim milk in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH7.5) buffer for 2 h at room temperature, then incubated with rabbit-anti-human NET-1 antibody (1:500 dilution, Abgent, USA), rabbit-anti-human EMS1 (1:500 dilution, Abcam, USA) and rabbit-anti-human VEGF antibody (1:500 dilution, Abcam, USA), mouse-anti-human β-actin antibody (1:500 dilution, Abcam, USA) as internal control. Then membranes were washed in TBST and incubated with a goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibody (1:1000 dilution, Jackson, USA) at room temperature for 2 h. At last, the specific proteins were detected with ECL chemiluminescence reagent (Beyotime, China); the membranes were exposed to films (Kodak, USA).
3
Quantification of SRPK1 Protein Levels
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The protein expression levels of SRPK1 in tissues or cells were detected by Western blot. Cells were plated into 6-well plates and grown for 24 hours, and posttreatment took 48 hours, as described earlier. The cells were then collected and lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific) on ice and centrifuged for collecting proteins. The proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Burlington, MA, USA). Subsequently, the membrane was incubated with rabbit anti-human SRPK1 antibody (1:1,000 dilution) or mouse anti-human β-actin antibody (1:5,000 dilution) (all from Abcam) as an internal control. After washing with TBST, the membrane was incubated with HRP-conjugated secondary antibody (1:5,000 dilution) (Abcam) for 1.5 hour at room temperature, then washed in TBST. The specific proteins were detected with ECL substrate (Thermo Fisher Scientific).
4
Quercetin Dihydrate Treatment Evaluation
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Quercetin dihydrate (Sigma, USA) was dissolved in dimethyl sulfoxide (DMSO; Corning, USA) and further diluted with medium to reach 0, 20, 40, 50, 80, 100, 150, 160, and 200 μM before treatment. The following primary antibodies were used in the present study: rabbit anti-human Fascin antibody (1:1000; Abcam, USA), rabbit anti-human Erzin antibody (1:1000; Cell Signaling Technology, USA), mouse anti-human MMP-2 antibody (1:1000; Cell Signaling Technology, USA), rabbit anti-human MMP-9 antibody (1:1000; Cell Signaling Technology, USA), mouse anti-human β-actin antibody (1:3000; Abcam, USA), rabbit anti-human GAPDH antibody (1:3000; Abcam, USA), mouse anti-human vimentin antibody (1:400; Proteintech, USA), and mouse anti-human cytokeratin antibody (1:400; Proteintech, USA). The secondary antibodies used in this study were listed as follows: peroxidase-conjugated goat anti-rabbit antibody (1:5000; Biosharp, China) and anti-mouse antibody (1:5000; Biosharp, China).
5
Protein Expression Analysis in BL-PDT-Treated Cells
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The expression levels of involved proteins were measured by western blotting analysis and analyzed by ImageJ software. Briefly, the cell samples at 24 h after BL-PDT were collected and lysed in 1 × radioimmunoprecipitation assay buffer with 1% phenylmethylsulfonyl fluoride for 20 min on ice. After quantification by a bicinchoninic acid assay kit (Beyotime, Shanghai, China), lysates were boiled for 10 min at 100 °C, and aliquots of 20 μg of protein were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes that were then blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies on a shaker (4 °C, overnight). After 3 times of washing with TBST and 2 h of incubation with the corresponding secondary antibody at room temperature, the immunoblots on the membranes were observed using an enhanced chemiluminescence detection kit (Millipore, Burlington, MA, USA). The primary antibodies included LC3 and PARP antibodies (all from Cell Signaling Technology, USA); p62, Beclin-1, Bax, Bcl-2, Caspase 3, and Caspase 9 antibodies (all from Abcam, UK); and PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR antibodies (all from Abmart, China). Mouse anti-human β-Actin antibody and goat anti-mouse or anti-rabbit secondary antibody were purchased from Abcam.
6
Western Blot Analysis of RACK1 Protein
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Western blot was performed according to the standard procedures. Briefly, tissues and cell lysates were extracted using RIPA buffer (Promega, USA), the protein concentrations were determined by a BCA™ Protein Assay Kit (Pierce, USA). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto PVDF membranes (Merk-Millipore, USA). The membranes were first incubated with indicated primary antibodies overnight (4 °C), and then followed by HRP-conjugated secondary antibodies for 2 h at room temperature. Blots were detected with ECL Western Blotting Substrate (Promega, USA) and quantized by Image J software (NIH, USA), β-actin was used as an internal control. The following antibodies were included: a rabbit anti-human RACK1 antibody (Abcam, USA, 1:500 dilution), a mouse anti-human β-actin antibody (Abcam, USA, 1:1,000 dilution), a horseradish peroxidase (HRP)-conjugated IgG (Abcam, USA, 1:2,000 dilution).
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