Cells were washed twice with PBS and total protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with PMSF protein inhibitor (Beyotime Institute of Biotechnology). The protein concentration was quantified using a standard BCA protein assay and 30 µg of protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes (MilliporeSigma) and blocked with 5% non-fat milk at 26˚C for 2 h. The membranes were then incubated overnight at 4˚C with the following primary antibodies: Rabbit anti-COPS7A (1:1,000; cat. no. ab124705; Abcam), rabbit anti-CDK2 (1:1,000; cat. no. ab32147; Abcam), rabbit anti-CDK4 (1:1,000; cat. no. ab108357; Abcam), rabbit anti-cyclin D2 (1:1,000; cat. no. ab230883; Abcam) and rabbit anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). Following the primary antibody incubation, the membranes were incubated with a goat anti-rabbit IgG H&L secondary antibody (1:5,000; cat. no. ab6721; Abcam) at room temperature for 2 h according to the protocol. The protein bands were visualized using an ECL system (Thermo Fisher Scientific, Inc.). GAPDH was used as the internal loading control.
Rabbit anti cdk2
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti-CDK2 is a primary antibody that recognizes the cyclin-dependent kinase 2 (CDK2) protein. CDK2 is a key regulator of the cell cycle and plays a crucial role in the G1/S transition. This antibody can be used to detect and study the expression and distribution of CDK2 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti cdk4, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Pmsf protein inhibitor, by Beyotime (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemmate, by Agilent Technologies (1 mentions)
Mouse anti cdk2, by Santa Cruz Biotechnology (1 mentions)
Dylight 488 donkey anti mouse iggs, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 555 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Cas block, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Guinea pig anti nephrin, by Progen Biotechnik (1 mentions)
Rabbit anti cyclin d1, by Abcam (1 mentions)
Antifade mounting medium with 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
Rabbit anti gapdh, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Yeasen (1 mentions)
Ecl western blot kit, by Biosharp (1 mentions)
5 protocols using rabbit anti cdk2
1
Western Blot Analysis of Cell Cycle Regulators
2
Immunofluorescence Staining of Podocytes and Mouse Kidney
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Differentiated human podocytes were fixed with 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS and permeabilized with 0.1% Triton X-100 in PBS. Podocytes were blocked with CAS-block (Invitrogen), stained with mouse anti-CDK2 (Santa Cruz Biotechnology) for one hour at RT in ChemMate (Dako Cytomation, Glostrup, Denmark), washed with PBS and incubated with DyLight 488 donkey anti-mouse IgG (Abcam) for one hour in ChemMate (Dako Cytomation). Perfused mouse kidney samples were fixed with 10% formaldehyde and embedded in paraffin. Deparaffinized sections were blocked with CAS-block (Invitrogen), incubated with rabbit anti-CDK2 (Abcam), guinea pig anti-nephrin (Progen Biotechnik GmbH, Heidelberg, Germany) or mouse anti-WT1 (Upstate, New York, USA) IgGs overnight at +4 °C in ChemMate (Dako Cytomation), washed with PBS and incubated with AlexaFluor 555 donkey anti-rabbit (Invitrogen), DyLight 488 donkey anti-quinea pig IgGs (Jackson Immuno Research Laboratories Inc., West Grove, PA) or DyLight 488 donkey anti-mouse IgGs (Jackson Immuno Research Laboratories Inc.). Samples were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and examined with Leica TCS CARS SP8 confocal microscope (Leica Microsystems CMS GmbH, Mannheim, Germany).
3
Investigating CED's Effects on NSCLC
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The human NSCLC cell line A549 (p53 wild-type) was provided by the Cell Bank of Chinese Academy of Sciences (Shanghai, China). CED (purity > 99.58%) was purchased from MedChemExpress (New Jersey, USA). CED was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at -80°C.
Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). Mouse anti-microtubule-associated protein B-light chain 3 (LC3B) was purchased from Cell Signaling Technology (Billerica, USA). Rabbit anti-GAPDH, mouse anti-mTOR, and rabbit anti-p-mTOR were purchased from Invitrogen (California, USA). Rabbit anti-VEGFR2, rabbit anti-VEGFR3, rabbit anti-P38, rabbit anti-p-P38, rabbit anti-Erk1/2, and rabbit anti-p-Erk1/2 were acquired from GeneTex (Taiwan, China). Rabbit Anti-CDK4, rabbit anti-cyclin D1, rabbit anti-CDK2, and rabbit anti-cyclin E were purchased from Abcam (Cambridge, England).
Cell counting kit-8 (CCK-8) was obtained from Yeasen Biotech (Shanghai, China). Antifade mounting medium with 4′,6-diamidino-2-phenylindole was obtained from Vector Laboratories (Shanghai, China). Annexin V-FITC/PI double staining was purchased from Becton, Dickinson and Company (New Jersey, USA).
Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). Mouse anti-microtubule-associated protein B-light chain 3 (LC3B) was purchased from Cell Signaling Technology (Billerica, USA). Rabbit anti-GAPDH, mouse anti-mTOR, and rabbit anti-p-mTOR were purchased from Invitrogen (California, USA). Rabbit anti-VEGFR2, rabbit anti-VEGFR3, rabbit anti-P38, rabbit anti-p-P38, rabbit anti-Erk1/2, and rabbit anti-p-Erk1/2 were acquired from GeneTex (Taiwan, China). Rabbit Anti-CDK4, rabbit anti-cyclin D1, rabbit anti-CDK2, and rabbit anti-cyclin E were purchased from Abcam (Cambridge, England).
Cell counting kit-8 (CCK-8) was obtained from Yeasen Biotech (Shanghai, China). Antifade mounting medium with 4′,6-diamidino-2-phenylindole was obtained from Vector Laboratories (Shanghai, China). Annexin V-FITC/PI double staining was purchased from Becton, Dickinson and Company (New Jersey, USA).
4
Protein Expression Analysis of DPCs
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After 48 h of transfection, the DPCs proteins were disposed of with RIPA lysis buffer (Beyotime, Shanghai, China), and concentrations were detected using the BCA method. The proteins were separated and then transferred to PVDF membranes, which were probed with 1:500 rabbit anti-EGR1 (Affinity, Melbourne, Australia), 1:2500 mouse anti-GAPDH (ABclonal, Wuhan, China), 1:1000 rabbit anti-PCNA (Abcam, Cambridge, UK), 1:1000 rabbit anti-CDK2 (Abcam, Cambridge, UK), 1:3000 goat anti-rabbit IgG HRG antibody (ABclon, Wuhan, China), and 1:3000 goat anti-mouse (ABclonal, Wuhan, China). The protein expressions were measured using the ECL Western Blot kit (BioSharp, Hefei, China), and analysed by the ChemiDocTM Analysis System (Bio-Rad, Hercules, CA, USA).
5
Immunostaining of Cultured Podocytes
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Podocytes cultured on black 96-well plates (PerkinElmer, Waltham, MA, USA) were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS and permeabilized with 0.1% Triton X-100 in PBS. Cells were incubated with rabbit anti-cleaved caspase-3 (Cell Signaling Technology), rabbit polyclonal anti-phospho-CDK2 (Cell Signaling Technology), or rabbit anti-CDK2 (Abcam) at room temperature for one hour, followed by IRDye 800 (LI-COR) donkey anti-rabbit IgG and 1 μM DRAQ5TM (Thermo Fisher Scientific, Waltham, MA, USA) at RT for one hour. Detection and quantification were performed with an Odyssey Infrared Imager (LI-COR). DRAQ5TM was used for normalization.
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