Western blotting revealed several functional proteins that directly affect cell migration, cell invasion 28 (link)-30 (link), and cell proliferation 31 (link), 32 (link). Cellular proteins were extracted using lysis buffer (P0013; Beyotime Biosciences, Shanghai, China) containing protease-inhibitor cocktail (B14002; Biotool, Shanghai, China). Proteins were quantified using the Bradford method. Approximately 60 µg of each protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), blocked in 5% skim milk (232100; Becton Dickenson, Franklin Lakes, NJ, USA) at 28 ºC, and incubated with antibodies (Table 1 ) overnight at 4 ºC. Membranes were then washed and incubated with horseradish peroxidase-conjugated anti-mouse/rabbit IgG (1:2000; ZSGB-BIO, Beijing, China) at 37 ºC for 2 h. Finally, immunoreactivity was detected using the BioImaging system (UVP, Inc., Upland, CA, USA).
Horseradish peroxidase conjugated anti mouse rabbit igg
Manufactured by ZSGB-BIO
Sourced in China
Horseradish peroxidase-conjugated anti-mouse/rabbit IgG is a secondary antibody that binds to mouse or rabbit primary antibodies. It is commonly used in immunoassays and immunohistochemistry techniques to detect and visualize target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
P0013, by Beyotime (2 mentions)
Bioimaging system, by Analytik Jena (1 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Gapdh, by Beyotime (1 mentions)
St506, by Beyotime (1 mentions)
St023, by Beyotime (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by Beyotime (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
4 protocols using horseradish peroxidase conjugated anti mouse rabbit igg
1
Western Blotting of Migration and Proliferation Proteins
2
Western Blot Analysis of Cellular Proteins
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Total cellular protein was extracted using the NP‐40 lysis buffer (P0013F; Beyotime Biosciences, Shanghai, China) containing a protease‐inhibitor PMSF (ST506; Beyotime Biosciences, Shanghai, China) and quantified using an enhanced BCA Protein Assay Kit (P0010; Beyotime Biosciences, Shanghai, China). Proteins (30 μg/lane) were resolved by 10% SDS‐PAGE gel, and transferred to 0.45 or 0.22 μm PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% BSA (st023; Beyotime Biosciences, Shanghai, China), and then incubated overnight at 4°C with the appropriate primary antibodies: anti‐TMEM107 (1:1000; Abcam; ab181396), E‐cadherin (1:500; cell signaling technology; 3195), N‐cadherin (1:500; Cell Signaling Technology; 13 116), vimentin (1:500; Cell Signaling Technology; 5741), MMP2 (1:500; Cell Signaling Technology; 87809), MMP9 (1:500; Cell Signaling Technology; 13667), and GAPDH (1:1000; Beyotime; AF0006). Membranes were washed with Tris‐buffered saline with Tween‐20 and then incubated with horseradish peroxidase‐conjugated anti‐mouse/rabbit IgG (1:2000; ZSGB‐BIO, Beijing, China) at room temperature for one hour. Target proteins were detected by ECL (Thermo Fisher Scientific, Waltham, MA, USA) using a BioImaging system (UVP, Inc., Upland, CA, USA).
3
Protein Expression Regulates Cell Migration
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The expression of proteins directly influences cell migration and invasion (15) (16) (17) (18) (19) as well as cell proliferation and cell cycle progression (20) (21) (22) (23) (24) . Total cellular or tissue protein was extracted using a lysis buffer (P0013, Beyotime, Biosciences, Shanghai, China) and added into a protease-inhibitor cocktail (B14002, Biotool, Shanghai, China) and phosphatase-inhibitor cocktail (B15002, Biotool).
Proteins (80 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gels), and were then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and the membranes were blocked with 5% skim milk (232100;Becton Dickenson, Franklin Lakes, NJ, USA) in Tris-buffered saline with Tween-20 at room temperature for 1 h. After being treated with the appropriate primary antibodies (Table 1) at 4℃ for 16 h, the proteins were treated with horseradish peroxidase-conjugated anti-mouse/rabbit IgG (1:200, ZSGB-BIO, Beijing, China) at 37℃ for 1 h. Immune reactivity was detected by enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) using a BioImaging system (UVP, Inc., Upland, CA,USA).
Relative protein expression was calculated after normalization to GAPDH or β-actin used as a loading control. The data were visualized and analyzed in ImageJ software.
Proteins (80 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gels), and were then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and the membranes were blocked with 5% skim milk (232100;Becton Dickenson, Franklin Lakes, NJ, USA) in Tris-buffered saline with Tween-20 at room temperature for 1 h. After being treated with the appropriate primary antibodies (Table 1) at 4℃ for 16 h, the proteins were treated with horseradish peroxidase-conjugated anti-mouse/rabbit IgG (1:200, ZSGB-BIO, Beijing, China) at 37℃ for 1 h. Immune reactivity was detected by enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) using a BioImaging system (UVP, Inc., Upland, CA,USA).
Relative protein expression was calculated after normalization to GAPDH or β-actin used as a loading control. The data were visualized and analyzed in ImageJ software.
4
Western Blotting Protein Analysis Protocol
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Transiently transfected cells were washed with ice-cold PBS and lysed using a lysis buffer (Beyotime, Shanghai, China) containing a protease-inhibitor cocktail (Beyotime) and a phosphatase-inhibitor cocktail (MedChemExpress). After quantitation using the bicinchoninic acid (BCA) protein assay, equal amounts of protein samples were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene difluoride membranes (Invitrogen). For Western blotting, the membranes were blocked with 5% skimmed dry milk solution in Tris-based saline-Tween 20 (TBS-T) at room temperature for 2 h and incubated with a primary antibody (Table 4) in 2%BSA in TBS-T at 4°C overnight. The next day, the membranes were washed with TBS-T thrice and incubated at 37 o C with horseradish peroxidase-conjugated anti-mouse/rabbit IgG at a dilution of 1:20000 (Zsbio, Beijing, China) for 2 h. After washing again with TBS-T, the membranes were incubated with the enhanced chemiluminescence reagent (Beyotime) to visualize the positive protein bands captured using an enhanced chemiluminescence device (Thermo Fisher Scientific, Waltham, MA USA). The results were quantified using ImageLab 3.0 software (Bio-Rad, Hercules, CA, USA).
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