Antigen retrieval on paraffin sections was performed using Trilogy (Cell Marque). After blocking, sections were incubated with primary antibodies overnight at 4°C, washed in PBS + 0.1% Tween 20 (PBST), and then incubated with secondary antibodies. Quantification was performed using ImageJ software. Primary antibodies used were anti-GFP (Invitrogen), anti-Ki67 (BD), anti-Flag (Sigma), anti-BrdU (BD), anti-YAP, anti-PH3, anti-CC3 (Cell Signaling Technology – data not shown), anti-MCM6, and anti-p21 (Santa Cruz Biotechnology). For immunoblotting, primary antibodies used were anti-YAP, anti-P-ERK, anti-ERK (Cell Signaling), anti-TEF1 (BD Biosciences), anti-p21, anti-E2F1, anti-Cyclin A, anti-PCNA, anti-MCM6 (Santa Cruz Biotechnology), and anti-Cyclin E (eBiosciences).
Anti p21
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom, Italy, Germany
Anti-p21 is a laboratory reagent used for the detection and analysis of the p21 protein in biological samples. p21 is a cyclin-dependent kinase inhibitor that plays a crucial role in cell cycle regulation. The Anti-p21 product is designed to enable researchers to study the expression and function of this important cellular protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti p53, by Santa Cruz Biotechnology (59 mentions)
Anti gapdh, by Santa Cruz Biotechnology (25 mentions)
Anti cyclin d1, by Santa Cruz Biotechnology (24 mentions)
Anti β actin, by Santa Cruz Biotechnology (23 mentions)
Anti actin, by Merck Group (16 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (15 mentions)
Anti β actin, by Merck Group (15 mentions)
Anti bax, by Santa Cruz Biotechnology (13 mentions)
Anti caspase 3, by Cell Signaling Technology (13 mentions)
Anti p27, by Santa Cruz Biotechnology (13 mentions)
Pvdf membrane, by Merck Group (13 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (11 mentions)
Anti β actin, by Cell Signaling Technology (10 mentions)
Anti cdk2, by Santa Cruz Biotechnology (10 mentions)
Anti parp, by Cell Signaling Technology (10 mentions)
Anti p16, by Santa Cruz Biotechnology (9 mentions)
Anti akt, by Cell Signaling Technology (8 mentions)
Anti actin, by Santa Cruz Biotechnology (8 mentions)
Anti cdk4, by Santa Cruz Biotechnology (8 mentions)
Anti cyclin e, by Santa Cruz Biotechnology (8 mentions)
199 protocols using anti p21
1
Immunohistochemistry and Immunoblotting Protocol
2
Western Blot and qPCR Analysis
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Radio-immunoprecipitation assay (RIPA) lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1.0 mM EDTA, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1% Triton X-100) was used to prepare cell or frozen tissue protein lysates. The protein concentration was determined by using BCA protein assay kit (Thermo) following the instructions. Protein lysates were resolved on 10% SDS‒PAGE gels using standard procedures. Anti-Sp1 (106 kDa; Santa Cruz), anti-Pax7 (44‒50 kDa; Santa Cruz), anti-Cyclin D1 (37 kDa; Santa Cruz), anti-MEF-2C (40‒65 kDa; Santa Cruz), anti-MyoG (34 kDa; Santa Cruz), anti-MyHC (200 kDa; Santa Cruz), anti-p21 (21 kDa; Santa Cruz), anti-Tubulin (55 kDa; Sigma Aldrich), anti-GAPDH (36 kDa; Sigma Aldrich), and anti-HSP90 (90 kDa; Cell Signaling Technology) were used for western blot analysis.
Trizol reagent (Thermo) was used to extract total RNA from cells or frozen tissues according to the standard procedure. First strand cDNA was synthesized by using the PrimeScript RT kit (TaKaRa). The miRNAs were reverse transcribed by stem-loop RT system, which was performed as described previously (Liu et al., 2014 (link)). qPCR was performed on an ABI7900 Real Time PCR System (Applied Biosystems). The detailed information of PCR primers is inSupplementary Table S1 .
Trizol reagent (Thermo) was used to extract total RNA from cells or frozen tissues according to the standard procedure. First strand cDNA was synthesized by using the PrimeScript RT kit (TaKaRa). The miRNAs were reverse transcribed by stem-loop RT system, which was performed as described previously (Liu et al., 2014 (link)). qPCR was performed on an ABI7900 Real Time PCR System (Applied Biosystems). The detailed information of PCR primers is in
3
Immunohistochemical Analysis of Inflammation in IVD
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Rat and human IVD tissues were obtained, fixed with 4% PFA for 48 h and then decalcified and embedded in paraffin. The tissues were cut into 5-μm-thick sections that were successively dewaxed and hydrated with xylene, anhydrous ethanol and an alcohol gradient (95%, 85% and 75%). The sections were then subjected to antigen retrieval with pepsin for 20 min at 37°C, incubated with neutralized endogenous peroxidase in a humidified box for 15‒20 min and sealed with goat serum for 30 min. The slides were then incubated overnight at 4°C with primary antibodies including anti-p-p65 (1:100; ABclonal), anti-HMGB1 (1:100; ABclonal), anti-NLRP3 (1:1000; Cell Signaling Technology), anti-caspase-1/p20 (1:100; Affinity), and anti-p21 (1:100; Santa Cruz Biotech, Santa Cruz, USA). The sections were subsequently rewarmed and incubated with biotin-labeled goat anti-mouse/rabbit secondary antibodies for 20‒30 min at room temperature and HRP-conjugated streptavidin (ZSGB-BIO, Beijing, China) for 25 min. DAB (ZSGB-BIO) staining and hematoxylin staining were performed for 1 min. Finally, images were captured using an Eclipse 80i fluorescence microscope (Nikon, Tyoko, Japan).
4
Investigating Receptor Signaling Pathways
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The following antibodies were used according to the protocols supplied by the manufacturers: anti-pERK1/2 (#9101, Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 (#4695, Cell Signaling Technology), anti-GAPDH (ENM0040, Elabscience Biotechnology Inc., Houston, TX, USA), anti-pEGFR (12A3) (sc-57542, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-EGFR (C-2) (sc-377229, Santa Cruz Biotechnology Inc.), anti-pAKT (#9271, Cell Signaling Technology), anti-AKT (#9272, Cell Signaling Technology), anti-Cyclin D1 (sc-753, Santa Cruz Biotechnology Inc.), anti-p21 (sc-397, Santa Cruz Biotechnology Inc.), and anti-beta Tubulin (Sigma), anti-alpha actin (Sigma). All the secondary antibodies (HRP-conjugated anti-rabbit and anti-mouse) were purchased from Santa Cruz Biotechnology Inc.
Drug formulations: gefitinib (ZD1839) and MG132 (S2619) were purchased from Selleckchem; AZD9291 was obtained from AstraZeneca; BAPTA_AM (HB0981) was from HelloBio.
Recombinant Human EGF (AF-100-15) was from PeproTech (London, UK).
Drug formulations: gefitinib (ZD1839) and MG132 (S2619) were purchased from Selleckchem; AZD9291 was obtained from AstraZeneca; BAPTA_AM (HB0981) was from HelloBio.
Recombinant Human EGF (AF-100-15) was from PeproTech (London, UK).
5
Canine Melanoma Cell Lines: Characterization and KPT-335 Study
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Canine melanoma cell lines Mel 23, Mel 36, Mel 69 and Mel 83 were generously provided by Michael S. Kent (UC Davis School of Veterinary Medicine, Davis, CA) [34 (link)-36 (link)]. Three of the lines (Mel 23, 69 and 83) were derived from a primary oral tumor and Mel 36 was generated from a metastatic lymph node. The cell lines were maintained in RPMI 1640 supplemented with 10% FBS, non-essential amino acids, sodium pyruvate, penicillin, streptomycin, L-glutamine, and Hepes (4-(2-hydroxythyl)-1-piperazineethanesolfonic acid) at 35°C, supplemented with 5% CO₂. KPT-335 (provided by Karyopharm Therapeutics, Inc, Natick, MA) was dissolved in DMSO to generate stock solutions for use in vitro. The following antibodies were used for Western blotting and immunofluorescence experiments: anti-p53, anti-p21, anti-XPO1, and anti- β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA).
6
Protein Expression Analysis of Cell Lines
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Exponentially growing cells were harvested and resuspended in PBS pH 7.4 (Gibco). Then the cells were lysed by lysis buffer mixed with protease inhibitor cocktail (Roche, Mannheim, Germany). The lysate was mixed with Laemmli sample buffer containing 5% 2-mercaptoethanol and boiled for 5 min. Equal amounts of protein extracts were separated on a 10% SDS/PAGE, blotted onto an activated polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) and blocked in TBST with 5% dried milk. Membranes were probed with anti-RAD51 (1:1000, Bioscience), anti-GAPDH (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-cyclin D1 (1:500, Santa Cruz Biotechnology), anti-p21 (1:500, Santa Cruz Biotechnology) at 4°C overnight. Then, they were probed with goat anti-mouse secondary antibodies (Thermo Fisher Scientific, Grand Island, NY, USA) for 1 h.
7
Immunohistochemical Analysis of HOXA5 Expression
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The technical process of immunohistochemistry (IHC) was performed as previously described7 (link). The HOXA5 staining strength was categorized into different groups depending on the positive cell percentage and positive cell staining density. The positive cell percentage was categorized into five grades: 0–3% (0), 3–25% (1), 26–50% (2), 51–75% (3), and 76–100% (4). The positive cell staining density was also categorized into four grades: negative (0), weak brown (1), moderate brown (2), and strong brown (3). The final immunohistochemical score was calculated as follows: immunoreactivity score (IRS) = intensity score × positive score. The final IRS were categorized into three groups: negative (≤3), weak positive (>3 but ≤6), and strong positive (>6). The antibodies used were as follows: anti-HOXA5 (1:100, sc-365784, Santa Cruz); anti-Ki67 (1:100, sc-23900, Santa Cruz); anti-cyclinD1 (1:100, sc-8396, Santa Cruz); anti-p21 (1:50, sc-817, Santa Cruz); anti-β-catenin (1:50, sc-7963, Santa Cruz); anti- p53 (1:50, sc-7963, Santa Cruz). The technical process of immunocytochemistry was performed as previously described22 (link).
8
Investigating SKA1 and Apoptosis Pathways
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TRIzol reagent and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The First Strand cDNA Synthesis kit and SYBR Premix Taq were from Takara (Dalian, Liaoning, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), bromodeoxyuridine (BrdU) and the anti-BrdU antibody were purchased from Sigma (St. Louis, MO, USA). DAPI, BCA protein assay and ECL Plus kits were obtained from Beyotime Institute of Biotechnology (Beijing, China). BD BioCoat Matrigel invasion chambers were purchased from BD Biosciences (San Jose, CA, USA).
The primary antibodies against human SKA1 and cleaved caspase-3 were obtained from Abcam (Cambridge, MA, USA). Anti-Bcl-2, anti-Bax, anti-p-ERK1/2, anti-ERK1/2, anti-p-Akt, anti-Akt, anti-p21, anti-cyclin D1 and anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology. Biotinylated- and Cy3-conjugated anti-rabbit secondary antibodies were purchased form Boster (Wuhan, Hubei, China).
The primary antibodies against human SKA1 and cleaved caspase-3 were obtained from Abcam (Cambridge, MA, USA). Anti-Bcl-2, anti-Bax, anti-p-ERK1/2, anti-ERK1/2, anti-p-Akt, anti-Akt, anti-p21, anti-cyclin D1 and anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology. Biotinylated- and Cy3-conjugated anti-rabbit secondary antibodies were purchased form Boster (Wuhan, Hubei, China).
9
Cytotoxicity Assessment of Chemotherapeutics
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CPT and cisplatin were obtained from Sigma; MTX, mitoxantrone, doxorubucin from Enzo Life Sciences, 5-fluorouridine from Acros Organics. The following antibodies were used: anti-p53 1:1,000 (Thermo Scientific, clone PAB240), anti-p21 1:1,000 (Santa Cruz, c-19), anti-γH2AX 1:2,500 (Millipore, JBW301), anti-phospho-ATM Ser1981 1:400 (Invitrogen, 10H11), anti-β-actin 1:10,000 (MP Biomedicals, C4).
10
Whole Cell Lysate Preparation and Immunoblotting
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Whole cell lysates were prepared in RIPA lysis buffer supplemented with protease inhibitor cocktail (Complete Mini; Roche, Basel, Switzerland). The lysates were sonicated for 5 minutes, centrifuged at 12 000 × g for 20 minutes at 4°C, and the supernatants were collected. Immunoblotting was carried out as previously described.29 The following primary antibodies were used at the indicated dilutions: anti‐β‐actin (1:10 000, A5441; Sigma‐Aldrich), anti‐p21 (1:1000, Sc‐397; Santa Cruz Biotechnology, Dallas, TX, USA), anti‐cleaved poly ADP‐ribose polymerase (PARP, 1:1000, #9541; Cell Signaling Technology, Danvers, MA, USA), anti‐IDH1 (1:200, 014‐24061; Wako), and anti‐IDH1‐R132S (1:200, 015‐24091; Wako).
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