PBS was used to wash fresh tissue which was grounded into a homogenate, and then RIPA buffer was used to lyse them to obtain total protein. Protein concentrations were determined by the BCA assay (Solarbio, PC0020, China). Samples (30 μg protein) were resolved on 8% SDS-PAGE gel and transferred to PVDF at 4°C. Membranes were blocked in fat-free milk combined with TBST for 1 hour at 25°C and immunoblotted with the appropriate diluted primary antibody KIF20A (Abcam, ab104118, UK; 1 : 1000 dilution for Western blot and 1 : 200 dilution for IHC-P), PCNA (Abcam, ab92552, UK; 1 : 1000 dilution for Western blot), Ki67 (Abcam, ab16667, UK; 1 : 1000 dilution for Western blot), MMP2 (Abcam, ab37150, UK; 1 : 500 dilution for Western blot), MMP9 (Abcam, ab76003, UK; 1 : 5000 dilution for Western blot), and GAPDH (SunGene Biotech, KM9002, China; 1 : 5000 dilution for Western blot) at 4°C overnight. Then, membranes were washed 10 minutes three times and incubated with the secondary antibody for 1 hour at 25°C. Finally, the blots were visualized by Imager.
Ab104118
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab104118 is a laboratory equipment product offered by Abcam. It is a tool designed to assist in scientific research and experiments, but a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Ab37150, by Abcam (2 mentions)
Ab92552, by Abcam (2 mentions)
Ab16667, by Abcam (2 mentions)
Km9002, by Sungene Biotech (2 mentions)
Ab76003, by Abcam (1 mentions)
Pc0020, by Solarbio (1 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Beyoecl plus reagent, by Beyotime (1 mentions)
Ab15580, by Abcam (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
3 protocols using ab104118
1
Protein Expression Analysis in Tissue Homogenate
2
Western Blot Analysis of Protein Expression
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Cells were washed with cold phosphate-buffered saline (PBS) and then homogenized using NP40 lysis buffer (Beyotime Biotechnology, Jiangsu, People’s Republic of China) containing protease inhibitors and phosphatase inhibitors. The total amount of proteins in cell lysates was quantified with Pierce™ bicinchoninic acid a protein assay kit (Thermo Fisher Scientific). After denatured with the sodium dodecyl sulfate (SDS) loading buffer and boiled for 5 min, ~30 μg proteins were loaded and separated by SDS-polyacrylamide gel electrophoresis and then electrotransferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat milk in Tris-buffered saline-Tween 20 followed by immunoblotting overnight at 4°C with primary antibodies (anti-KIF20A, Abcam, #ab104118; anti-Ki67, Abcam, #ab15580; anti-glyceraldehyde 3-phosphate dehydrogenase [GAPDH], Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-47724, 1:1,000). After being washed with Tris-buffered saline-Tween 20 and incubated with the secondary antibodies for another 1 h, the immunoreactivity was detected with the BeyoECL Plus reagent (Beyotime Biotechnology).
3
Antibody Validation for Protein Analysis
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Antibodies for the following proteins were used in this study for western blot and immunohistochemistry: KIF20A (Abcam, ab104118, 1 : 1000 dilution for western blot and 1 : 200 dilution for IHC-P), PCNA (Abcam, ab92552, 1 : 1000 dilution for western blot), Ki67 (Abcam, ab16667, 1 : 1000 dilution for western blot), Bcl-2 (Abcam, ab32124, 1 : 1000 dilution for western blot), caspase-3 (Abcam, ab13847, 1 : 500 dilution for western blot), MMP-2 (Abcam, ab37150, 1 : 500 dilution for western blot), and GAPDH (Sungene Biotech, KM9002, 1 : 5000 dilution for western blot).
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