Anti-CDK4 antibody was purchased from Proteintech (Rosemont, USA) and horseradish peroxidase (HRP)-conjugated Goat anti-Rabbit IgG, HRP-conjugated Goat anti-Mouse IgG, and anti-β-actin antibodies were purchased from Cell Signaling Technology (Beverly, USA). RIPA lysis buffer was purchased from Solarbio Life Sciences (Beijing, China). Briefly, cell samples or tissue samples were collected and lysed using RIPA reagent supplemented with phenylmethylsulfonyl fluoride (Roche) and a protease inhibitor cocktail (Roche, Pleasanton, CA, USA). The concentrations of all protein samples were detected using a BCA Protein assay kit (Beyotime). Equal amounts of protein extracts were added to gel wells and separated by 10% SDS-polyacrylamide gel electrophoresis gels. After electrophoresis and transferring protein bands to polyvinylidene fluoride membranes, the membranes were blocked for 1 h with 5% non-fat milk dissolved in Tris-buffered saline and incubated with primary antibodies at 4 °C for more than 12 h. The HRP-conjugated secondary antibodies were used and antigen–antibody complexes were tested by enhanced chemiluminescence system (Pierce, Rockford, IL, USA) according to the directions of the manufacturer.
Anti cdk4 antibody
Manufactured by Proteintech
Sourced in United States, China
The Anti-CDK4 antibody is a laboratory reagent used for the detection and analysis of the CDK4 protein, a key regulator of the cell cycle. This antibody is designed for use in various immunological techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to help researchers study the expression and function of CDK4 in biological samples.
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Lab products found in correlation
Phenylmethylsulfonyl fluoride, by Roche (1 mentions)
Horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti mouse igg, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Anti p53, by Agilent Technologies (1 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Dmi3000b inverted microscope, by Leica (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
5 protocols using anti cdk4 antibody
1
Western Blot Analysis Protocol
2
Immunohistochemical Analysis of DLL3, BID, CDK4, and P53
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Formalin-fixed tissue paraffin block made 3 mm white plate for IHC analysis. We used anti-DLL3 antibody (1:200; Proteintech), anti-BID antibody (1:200; Proteintech), anti-CDK4 antibody (1:200; Proteintech) and anti-P53 (1:100; Dako) for tissue staining. Three pathologists used a semi-quantitative immune response scoring algorithm to evaluate DLL3 staining independently. The semiquantitative immunoreactivity score (IRS) is between 0 and 30, based on the increase in IHC staining intensity (0, negative; 1, weak; 2, middle; 3, strong) and the percentage of positive tumor cells (one point per 10% increase, the percentage of positive tumor cells 1–10) [19 (link)]. The critical value of p53 staining was 10% (≤ 10% of tumor cells were negative and > 10% were positive) [20 (link),21 (link)]. Disagreements were resolved by three pathologists negotiated under a multi- lens.
Wilcoxon test and paired analysis between normal tissue and tumor tissue in the same patient were used to analyze the expression of DLL3. Kaplan-Meier curve was performed to analyze the survival of DLL3.
Wilcoxon test and paired analysis between normal tissue and tumor tissue in the same patient were used to analyze the expression of DLL3. Kaplan-Meier curve was performed to analyze the survival of DLL3.
3
Quantifying Protein Expression via Western Blot
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The transfected cells were collected, total protein was extracted, and the protein concentration was quantified using the BCA Protein Assay Kit. Then it was incubated with anti-CDK4 antibody (1:1000, Proteintech, Chicago, IL, USA) and anti-GAPDH antibody (1:1000, Abcam, Cambridge, UK) overnight at 4°C. Then anti-rabbit secondary antibody (1:1000, Cell Signaling Technology, Boston, MA, USA) was added to incubate for 1 h. GAPDH was used as an internal reference, and relative protein expression was expressed as the ratio of the gray value of the target band/GAPDH band. The specific experimental methods for Western blot analysis were performed with reference to the literature.29 (link)
4
Tumor Xenograft Experiment in Nude Mice
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BALB/c nude male mice (4 weeks old) were obtained from the Laboratory Animal Department of Central South University (Changsha, P.R. China) and maintained under specific pathogen-free conditions. This animal study was approved by the Institutional Animal Ethics Committee of Central South University (project identification code: 2018sydw0111, date of approval: April 12, 2018). For the tumor xenograft experiments, nude mice were randomly divided into two groups (n = 5/group), including the sh-NC group and sh-1 group. Lentiviral-infected tumor cells (5 × 106 cells) were resuspended in 100 μL of free culture medium and injected subcutaneously into the right and left flanks of the nude mice. The tumor width (W) and length (L) were monitored twice per week, and the tumor volume was calculated using the following formula: V = 1/2 (L × W2). Four weeks after the injection, the mice were killed by cervical dislocation, and the tumors were isolated, weighed, and immersed in formalin.
Next, the tumors were embedded in paraffin and cut into 4.0-μm sections for H&E staining and IHC. H&E staining and IHC were performed according to described protocols.60 (link),61 (link) Anti-Ki-67 antibody (Beijing Zsbio, P.R. China) and anti-CDK4 antibody (Proteintech, P.R. China) were used for IHC assay. Images were captured using a Leica DMI3000B inverted microscope (Germany).
Next, the tumors were embedded in paraffin and cut into 4.0-μm sections for H&E staining and IHC. H&E staining and IHC were performed according to described protocols.60 (link),61 (link) Anti-Ki-67 antibody (Beijing Zsbio, P.R. China) and anti-CDK4 antibody (Proteintech, P.R. China) were used for IHC assay. Images were captured using a Leica DMI3000B inverted microscope (Germany).
5
RIP Assay for CDK4 Binding
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RIP assays were performed using the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, USA). Anti-CDK4 antibody (Proteintech, P.R. China) and rabbit IgG purified (Millipore, USA) were used for RIP assay.62 (link) In brief, 2 × 107 cells were lysed in 0.1 mL of complete RIP lysis buffer containing RIP lysis buffer with protease inhibitors and RNase Inhibitor, and then centrifuged at 14,000 rpm for 10 min at 4°C. The supernatants and magnetic beads were incubated with 5 μg of CDK4 antibody or negative control rabbit IgG at 4°C overnight with gentle rotation. The beads were washed six times with ice-cold RIP wash buffer. Then, the RNA was purified, and quantitative real-time PCR was performed as described above.
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