According to the manufacturer’s protocol, RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) was employed to extract protein from cells. Protein concentrations were measured using a BCA Protein Assay Kit obtained from Beyotime Institute of Biotechnology Co., Ltd. (Shanghai, China). Next, 10% dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to separate the proteins, which were subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore) through electro-blotting. Then, 5% skim milk was used to block the membranes for 1 hour in tris-buffered saline with Tween (TBST), after which the membranes were incubated with primary antibody rabbit anti-ATG7 antibody (1:1,000, #8558, Cell Signaling), anti-p21 antibody (1:1,000, #2947, Cell Signaling), anti-Survivin (1:1,000, #2808, Cell Signaling), anti-E-cadherin (1:1,000, #3195, Cell Signaling), N-cadherin (1:1,000, #13116, Cell Signaling), VEGF (1:1,000, #9698, Cell Signaling), and Vimentin (1:1,000, #5741, Cell Signaling). After incubation overnight at 4 °C, the membranes were incubated for a second time with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies for 1 hour at 37 °C. The band densities were analyzed with an automatic digital gel image analysis system (Bio-Rad CFX-96, CA, USA).
Anti p21 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-p21 antibody is a laboratory research tool used to detect and analyze the p21 protein, which is involved in cell cycle regulation. This antibody can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of the p21 protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Anti β actin antibody, by Cell Signaling Technology (2 mentions)
Anti trf2 antibody, by Novus Biologicals (2 mentions)
Immobilon fl, by Merck Group (2 mentions)
Anti mouse and anti rabbit alkaline phosphatase conjugates, by Merck Group (2 mentions)
Anti gapdh antibody, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Vascular endothelial growth factor (vegf), by Cell Signaling Technology (1 mentions)
Anti survivin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Ab94396, by Abcam (1 mentions)
Ab5535, by Abcam (1 mentions)
Alexa 405, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l alexa fluor 405, by Abcam (1 mentions)
19 protocols using anti p21 antibody
1
Western Blot Analysis of Protein Markers
2
Immunostaining of Chondrocyte Transcription Factors
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Primary cell cultures were fixed for 15 min at room temperature with 3% formaldehyde/PBS. The fixed cultures were washed and permeabilized with 0.1% Triton X‐100/PBS at room temperature for 10 min, incubated with primary antibodies diluted in PBS at room temperature for 1 hr, washed with PBS, and then incubated with regular secondary antibodies conjugated to Alexa 405 (Life Technologies) for 1 hr at room temperature. The following primary and secondary antibodies were used: Anti‐SOX9 antibody (Merck Millipore, AB5535), Anti‐SOX5 antibody (Abcam, ab94396), Anti‐SOX6 antibody (Abcam, ab30455), Anti‐p21 antibody (Cell Signaling Technology, 2946), Goat anti‐Rabbit IgG H + L Alexa Fluor 405 (Thermo Fisher, A‐31556), Goat anti‐mouse IgG H + L Alexa Fluor 405 (Abcam, ab175660).
3
Western Blot Analysis of Cell Lysates
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Cells were lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime, China), and protein was harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, USA). After the incubation with a high affinity anti-p21 antibody (1:1000), anti-PTEN antibody (1:1000), anti-β-actin antibody (1:2000) (Cell Signaling Technology, USA), the membranes were then incubated with a secondary antibody (1:5000, Cell Signaling Technology, USA). After washes, signals were detected using a chemiluminescence system (Bio-Rad, USA) and analyzed using Image Lab Software.
4
Western Blot Analysis of Cellular Proteins
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For western blot analysis, protein lysates were prepared by suspending cell pellets in 1X cell culture lysis buffer (Promega). Protein was separated using 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon FL, Millipore). Primary antibodies- anti-TRF2 antibody (Novus Biological), anti-p21antibody (Cell signaling technology) and anti-β-actin/anti-GAPDH antibody (Sigma), anti-H2A (abcam). Secondary antibodies, anti-mouse and anti-rabbit alkaline phosphatase conjugates were from Sigma. The blot was finally developed by using Thermo Scientific Pierce NBT/BCIP developing reagents.
5
Western Blot Analysis of Protein Expression
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Total proteins in cells were collected using cell lysis buffer (Beyotime Biotechnology). Bradford Protein Assay Kit (Beyotime Biotechnology) was used for measuring proteins concentration. Western blotting was conducted using a similar method described in the earlier literature. Anti-proliferating cell nuclear antigen (PCNA) antibody (#13110), anti-p21 antibody (#2947), anti-SOX2 antibody (#3579), anti-N-cadherin (N-cad) antibody (#13116), anti-P65 antibody (#8242), anti-P65S536 antibody (#3033), anti-TAK1 (#5206), anti-phospho-IKKβ (ser180, #2697), anti-IKKβ (#8943) and anti-GAPDH antibody (#5174) were supplied by Cell Signaling Technology (MA, USA). Anti-TRAF6 antibody (ab33915), anti-vascular cell adhesion molecule-1 (VCAM1) antibody (ab134047), anti-CD44 antibody (ab189524) and anti-matrix metalloproteinase 14 (MMP14) antibody (ab51074) were supplied by Abcam Biotechnology (MA, USA). Signals of proteins were visualized through enhanced chemiluminescence technique. The relative protein expressions were calculated using Image J software.
6
Evaluating Cellular Stress Responses
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Endothelial cells were seeded at the density of 50.000/well in 24 multiwell plates in medium with 10% serum. After cell adhesion, NPs or lyophilized biomaterials were dissolved in medium and placed in direct contact with the cells. After 24 h incubation, biomaterials were gently removed and cells were lysed as described in [20 (link)]. Cell lysates were centrifuged at 10000 g for 20 min at 4°C. Following gel electrophoresis, proteins were blotted onto activated nitrocellulose membranes, incubated overnight with the anti caspase-3 antibody (1:1000) (Cell Signaling, Danvers, USA), anti p21 antibody (1:1000, Cell Signaling), anti p53 antibody (1:1000) (Santa Cruz, Heidelberg, Germany), anti inducible nitric oxide synthase (iNOS, 1:1000, Santa Cruz) or anti cyclooxygenase-2 (COX-2) antibody (1:1000) (Cayman, Ann Arbor, USA). The primary antibody was detected by incubating the membranes for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (Promega, Madison, USA)) diluted 1:2500 in PBS, followed by enhanced chemiluminescence detection system (Bio-Rad, Hercules, USA). Images were digitalized with CHEMI DOC Quantity One. Results were normalized to those obtained by using an antibody anti β-actin (1:10000) (Sigma). Original blots are reported as Supplemental Information S1 Fig .
7
Immunofluorescence Staining of Senescent HUVECs
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We seeded HUVECs on 24-well plates, fixed them with 4% PFA, and incubated them with anti-p16 antibodies (1:200, Cat# 10883-1-AP, Proteintech), anti-p21 antibody (1:200, Cat# 2947, Cell Signaling Technology, USA), anti-Lamp1 antibody (1:200, Cat# 9091, Cell Signaling Technology), and anti-p62 antibody (1:200, Cat# 88,588, Cell Signaling Technology) overnight at 4 °C and secondary antibodies (1:1000; Cat# A11012, Cat# 11,001; Invitrogen) at room temperature, with DAPI staining nuclei for 10 min, and the cells were taken by inverted fluorescence microscopy.
8
Western Blot Analysis of Cell Lysates
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Cells cultured in 75 cm2 flasks were washed three times with PBS and lysed with liquid nitrogen in a lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) with a protease inhibitor cocktail (Sigma-Aldrich, USA), followed by centrifugation at 1,000 ×g for 5 min. The protein content was quantified by Lowry et al. (1951) (link) and stored at −80°C (Lowry et al., 1951 (link)). An amount of 150 μg of the cell lysates was run on 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride low-fluorescence membranes (Millipore). The membranes were blocked with LI-COR blocking buffer for 1 h at 4°C and incubated overnight with anti-p53 antibody (1:10,000) (DO-1, Santa Cruz Biotechnology), anti-p21 antibody (Cell Signaling), or anti-MDM2 antibody (Cell Signaling), and then followed by IR-Dye® 800CW goat anti-mouse antibody (LI-COR) (1:15,000) for 1 h at room temperature. β-Actin was used as a loading control (Sigma-Aldrich). Signal detection was carried out using the Odyssey detection system (LI-COR), according to the manufacturer’s instructions. The quantification of band intensity was performed with ImageJ software (version 1.43r, National Institutes of Health) and normalized against HepG2, a WT-expressing cell line.
9
Western Blot Analysis of Cellular Proteins
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For western blot analysis, protein lysates were prepared by resuspending cell pellets in passive lysis buffer. Protein was separated using 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon FL, Millipore). After blocking the membrane was incubated with primary antibodies- anti-TRF2 antibody (Novus Biological), anti-p21antibody (Cell Signaling Technology), anti-TRF1 (Novus Biological), anti-gapdh (Santacruz biotechnology) and anti-β-actin antibody (Sigma). Secondary antibodies, anti-mouse and anti-rabbit alkaline phosphatase conjugates were from Sigma. The blot was finally developed by using Thermo Scientific Pierce NBT/BCIP developing reagents.
10
Protein Expression Analysis by Western Blot
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Total protein was extracted with RIPA buffer and quantified with bicinchoninic acid (BCA) protein quantitative assay (Thermo, USA). The samples were separated by 10% SDS-PAGE and transferred into PVDF membranes (Merck Millipore, USA). The membranes were blocked in 3% Bovine Serum Albumin (BSA) with PBS. Then, the membranes were incubated with corresponding antibodies: anti-ADSL antibody (Abcam, United Kingdom), anti-β-actin antibody (Cell Signaling Technology, USA), anti-Rb antibody (Cell Signaling Technology), anti-p21 antibody (Cell Signaling Technology), anti-CDK4 antibody (Cell Signaling Technology), anti-CDC2 antibody (Cell Signaling Technology), anti-Bcl2 antibody (Abcam), anti-Bax antibody (Abcam), anti-p27 antibody (Cell Signaling Technology), anti-Bid antibody (Cell Signaling Technology), anti-Bim antibody (Cell Signaling Technology). Appropriate second antibodies were applied in the next incubation. Lastly, the enhanced chemiluminescence (ECL) detection system (ImageQuant LAS 500, USA) was used to detect the membranes following the manufacturer’s protocol.
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