Ki 67

Manufactured by Wanlei

Sourced in China

The Ki-67 is a protein that is present during all active phases of the cell cycle, but is absent from resting cells. It is commonly used as a marker to determine the growth fraction of a given cell population.

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Lab products found in correlation

Pvdf membrane, by Merck Group (2 mentions) P jak2, by Affinity Biosciences (1 mentions) Interleukin 6 (il 6), by Wanlei (1 mentions) Mpm 2, by Abcam (1 mentions) P akt, by Cell Signaling Technology (1 mentions) Cell cycle analysis kit, by Beyotime (1 mentions) β actin, by Affinity Biosciences (1 mentions) Gapdh, by Affinity Biosciences (1 mentions) Cell counting kit 8, by Beyotime (1 mentions) Cpt1a, by Proteintech (1 mentions) Pdvf membrane, by Merck Group (1 mentions) Ecl reagent, by Beyotime (1 mentions) Chemidoc xrs system, by Bio-Rad (1 mentions) Gapdh, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Roche (1 mentions) Cleaved caspase 3, by Wanlei (1 mentions) Envision hrp rb, by Agilent Technologies (1 mentions) Te 2000u camera, by Nikon (1 mentions) Proliferating cell nuclear antigen (pcna), by Proteintech (1 mentions)

6 protocols using ki 67

Gastric Cancer Cell Lines: Culture and Evaluation

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Human gastric cancer cell lines HGC-27 and AGS were purchased from Procell Life Science and Technology Ltd. Gastric cancer cells HGC-27 and AGS were cultured in Dulbecco's modified Eagle's medium (DMEM) and RPMI 1640, respectively, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution in a 37°C cell incubator with 5% CO₂.
DMEM and RPMI 1640 medium were purchased from HyClone (USA). TAS-102, FTD and CTS were obtained from TargetMol (China), and their purity was greater than 98%. Cell Counting Kit (CCK)-8 and cell cycle analysis kit were obtained from Beyotime (China), and Annexin V-FITC/PI double staining cell apoptosis detection kit was purchased from KeyGEN BioTHCE (China). Primary antibodies included BrdU, Cyclin B1, Cyclin D1, Caspase 3, Caspase 9, Bcl-2, Survivin, phospho-extracellular signal-regulated kinase (p-ERK) (Thr202/Tyr204) and ERK, p-AKT (Ser473) and AKT, p-STAT3 (Tyr705) and STAT3 (Cell Signaling Technology), p-JAK2 (Tyr221) and JAK2, GAPDH, β-actin (Affinity), MPM-2 (Abcam), IL-6 and Ki-67 (Wanleibio). Secondary antibodies horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse IgG and Cy3 labeled goat anti-mouse IgG were from ZSGB-Bio (China) and Abclonal (China), respectively.

Luo P.Q., Zhang L.X., Chen Z.M., Wang G., Zhu H., Ying S., Wei Z.J., Han W.X, & Xu A.M. (2023). Effects and mechanisms of trifluridine alone or in combination with cryptotanshinone in inhibiting malignant biological behavior of gastric cancer. Cell Cycle, 22(12), 1463-1477.

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Protein extraction and Western blot analysis

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The total protein was extracted using cell lysis buffer. Protein samples were separated by 6−10 % SDS-PAGE and then blotted on a PDVF membrane (Millipore). Membranes were blocked in 5 % non-fat milk diluted by TBST and incubated the following antibodies overnight at 4 °C:DEC2 (1:1000, Proteintech), NR2F1 (1:1000, Proteintech), p27 (1:500, HUABIO), Ki67(1:500,Wanleibio), FASN (1:1000, Proteintech), CPT1A(1:1000, Proteintech), DGAT1(1:1000, Zenbio), DGAT2 (1:500 Cohesion Bioscience), HMGCR(1:1000, Zenbio), ACAT1(1:1000, Zenbio), GAPDH (1:5000, SAB signaling). After incubating anti-rabbit or anti-mouse secondary antibody, immunoblots were visualized by using chemiliminescence (ECL) reagent (Beyotime Biotechnology) and ChemiDoc XRS+System (Bio-Rad).

Dai L., Xian H., Wang H., Li M., Zhang M., Liang X.H, & Tang Y.L. (2023). Hypoxia induced cell dormancy of salivary adenoid cystic carcinoma through miR-922/DEC2 axis. Translational Oncology, 40, 101868.

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Protein Expression Analysis Protocol

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48h after transfection, cells were collected and lysed using a RIPA lysis buffer that contained the protease inhibitor cocktail (Roche, Basel, Switzerland) and phenyl methyl sulfonyl fluoride (PMSF) (Roche). Cell protein lysates that contained 50μg protein were electrophoresed on 10% or 12% sodium dodecyl sulfate-polyacrylamide gel, and then transferred onto 0.22mm or 0.45mm PVDF membrane (Millipore, Bedford, MA). Then, the membranes were incubated in blocking solution (5% non-fat milk) for 1.5 hour at room temperature (RT) and incubated with specific primary antibodies at 4°C overnight. The following antibodies were used in this study: GAPDH (Sigma, G9545); ACTB (Beyotime, AA128); Cleaved Caspase-3 (Wanleibio, WL02348); Ki-67 (Wanleibio, WL01384a); GADD45A (Sigma, WH00016 47M1).

Li L., Yin J.Y., He F.Z., Huang M.S., Zhu T., Gao Y.F., Chen Y.X., Zhou D.B., Chen X., Sun L.Q., Zhang W., Zhou H.H, & Liu Z.Q. (2017). Long noncoding RNA SFTA1P promoted apoptosis and increased cisplatin chemosensitivity via regulating the hnRNP-U-GADD45A axis in lung squamous cell carcinoma. Oncotarget, 8(57), 97476-97489.

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Immunohistochemical Analysis of Tumor Tissue

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Tumor segments were fixed with 4% paraformaldehyde overnight at 4°C and subsequently embedded in paraffin. The segments were then sectioned to a thickness of 4 μm. Dewaxing procedures were followed by blocking non‐specific sites with a 5% BSA solution for 1 h. Subsequently, the sections were incubated overnight at 4°C with primary antibodies against Ki‐67 (Wanleibio, Shenyang, China) or PLIN3 (Proteintech, Wuhan, China) and washed with PBS. EnVision+/HRP/Rb (DAKO, Glostrup, Denmark) was applied for 1 h at 37°C. The segments were then covered with 3,3′‐Diaminobenzidine (3,3′‐DAB, Maxim, Fuzhou, China) for 5 min and counterstained with hematoxylin for 30 s. Finally, sections were photographed with a TE2000‐U camera (Nikon, Tokyo, Japan).

Liang W., Zhou Z., Gao Q., Zhu Z., Zhu J., Lin J., Wen Y., Qian F., Wang L., Zhai Y., Lv J., Zhang H., Zhong F, & Du H. (2024). Tumor‐derived Prevotella intermedia aggravates gastric cancer by enhancing Perilipin 3 expression. Cancer Science, 115(4), 1141-1153.

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Western Blot Analysis of Protein Expression

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Proteins were extracted using RIPA buffer (Solarbio, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride (Solarbio). Protein concentration was determined using the Bradford method (Solarbio). Equal amounts of proteins were separated by SDS-PAGE and transferred onto a PVDF membrane (Merck, Germany). After blocking with 5% non-fat milk in TBST for 2 h, the membranes were incubated with primary antibodies against CCND2 (1:500, Proteintech, Wuhan, China), PCNA (1:1,000, Proteintech), Ki-67 (1:500, Wanleibio, Shenyang, China), p-ERK1/2 (1:300, Wanleibio), p-p38 (1:750, Wanleibio), p-JNK (1:300, Wanleibio), MMP9 (1:1,000, Wanleibio), MMP2 (1:500, Wanleibio), α-SMA (1:500, Wanleibio), GAPDH (1:1,000, Proteintech) and β-tubulin (1:1,000, Proteintech) at 4°C overnight. After washing with TBST, the membranes were then incubated with goat anti-rabbit IgG secondary antibody (dilution at a 1:20,000, Wanleibio) at 37°C for 2 h. Signal was visualized by ChemiDoc^TM MP Imaging System (BIO-RAD, California, USA). The experiment was repeated at least three times.

Lin J.J., Chen W., Gong M., Xu X., Du M.Y., Wang S.F., Yang L.Y., Wang Y., Liu K.X., Kong P., Li B., Liu K., Li Y.M., Dong L.H, & Sun S.G. (2021). Expression and Functional Analysis of lncRNAs Involved in Platelet-Derived Growth Factor-BB-Induced Proliferation of Human Aortic Smooth Muscle Cells. Frontiers in Cardiovascular Medicine, 8, 702718.

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Immunohistochemical Analysis of Colon Tissue

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Paraffin-embedded slides from colon tissue were firstly deparaffinized using dimethyl benzene and graded ethanol, then blocked with 5% bovine serum albumin (BSA), and stained with primary antibodies overnight at 4°C as follows: Ki67, F4/80, and proliferating cell nuclear antigen (PCNA) (1:200; Wanlei Biotechnology, Liaoning, China). This was followed by staining with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibodies for 20 min. Sections were dyed with diaminobenzidine (DAB) substrate color liquid after washing. Images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA).

Wang D., Zheng Y., Fan Y., He Y., Liu K., Deng S, & Liu Y. (2023). Sodium Humate-Derived Gut Microbiota Ameliorates Intestinal Dysfunction Induced by Salmonella Typhimurium in Mice. Microbiology Spectrum, 11(3), e05348-22.

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