RIPA (GBCBIO, G3424) was used to lyse cells and extract total protein to prepare protein samples. The protein lysates were separated by conventional electrophoresis, transferred onto the membrane, and washed with PBS, followed by blocking with 1% BSA for 2 h. Then, primary antibodies Box (ab32503, Abcam, Cambridge, UK), Bcl-2 (ab32124, Abcam, Cambridge, UK), Caspase-1 (ab32503, Abcam, Cambridge, UK), Cleaved Caspase-3 (ab32503, Abcam, Cambridge, UK), Caspase-8 (ab207802, Abcam, Cambridge, UK), Caspase-9 (ab2302, Abcam, Cambridge, UK), Caspase-11 (ab25901, Abcam, Cambridge, UK), Cyclin D1 (ab202068, Abcam, Cambridge, UK), ERK 1/2 (ab180673, Abcam, Cambridge, UK), p-ERK 1/2 (ab16663, Abcam, Cambridge, UK), WNT (ab17942, Abcam, Cambridge, UK), β-catenin (ab223500, Abcam, Cambridge, UK), GSDMD (ab15251, Abcam, Cambridge, UK) and β-actin (ab32572, Abcam, Cambridge, UK) (abcam, ab32503, ab32124, ab207802, ab2302, ab25901, ab202068, ab180673, ab16663, ab17942, ab223500, ab15251, ab32572, ab219800, ab8227) each at 1:1000 dilution were incubated with the membrane overnight at 4°C. The next day, HRP (ab32572, Abcam, Cambridge, UK) and labeled IgG secondary antibody (ab150077, Abcam, Cambridge, UK) each at 1:5,000 were further incubated with the membrane at room temperature for 1 h. ECL Luminescence Kit (Thermo, 32209) was used to develop the signal in a dark room.
Ab2302
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab2302 is a laboratory consumable product manufactured by Abcam. It is designed to facilitate specific research applications, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information about the intended use of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (88 mentions)
Ab32124, by Abcam (51 mentions)
Ab205718, by Abcam (42 mentions)
Pvdf membrane, by Merck Group (40 mentions)
Ab2324, by Abcam (39 mentions)
Ab8245, by Abcam (35 mentions)
Ab181602, by Abcam (32 mentions)
Ripa lysis buffer, by Beyotime (29 mentions)
Ab13847, by Abcam (29 mentions)
Ab9485, by Abcam (27 mentions)
Ab182858, by Abcam (21 mentions)
Ripa buffer, by Beyotime (19 mentions)
Ab59348, by Abcam (19 mentions)
Ab6721, by Abcam (19 mentions)
Ab8227, by Abcam (18 mentions)
Ab196495, by Abcam (17 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Ab205719, by Abcam (13 mentions)
Ab32064, by Abcam (13 mentions)
Bca protein assay kit, by Beyotime (13 mentions)
369 protocols using ab2302
1
Western Blot Analysis of Apoptosis Markers
2
Immunohistochemical Visualization of Apoptosis and Dopamine Markers
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To eliminate endogenous peroxidase activity prior to immunohistochemical staining, prefrontal cortical (PFC) tissue sections were treated with 3% hydrogen peroxide. For antigen retrieval, the slides were rinsed three times in PBS (pH 7.4) and then inoculated in sodium citrate buffer (0.01 M, pH 6.0) for 30 minutes in a water bath (95 °C). The slides were inoculated with bovine serum albumin (BSA) (1%, 1 hour) after reaching room temperature, and subsequently with the principal antibodies (4 C, overnight): anti-caspase-3 (ab2302, Abcam- Cambridge, UK, 1:100) (ab2302, Abcam- Cambridge, UK, 1:100) (23 (link)) or recombinant anti-tyrosine hydroxylase antibody (ab137869, Abcam, Cambridge-UK, 1:200) (24 (link)). Secondary antibodies conjugated with horseradish peroxidase were incubated on the sections for 0.5 hours at 37 °C, followed by labeled streptavidin-biotin (0.5 h) (DETHP1000, Sigma-Aldrich). Finally, we used hematoxylin as a counterstain and diaminobenzidine (DAB: 3 min) to envision the reaction.
3
Osteosarcoma Cell Line Analysis
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The following reagents and antibodies were used in this study: hFOB cells (CL‐0353, Procell), 143B cells (CRL‐8303, ATCC), MG‐63 cells (CL‐0157, Procell), Saos‐2 cells (CL‐0202, Procell), and HOS cells (CL‐0360, Procell). Anti‐COLEC12 (ab81136; Abcam), anti‐BAX (ab32503; Abcam), anti‐BCL‐2 (ab182858; Abcam), anti‐cleaved caspase‐3 (ab2302; Abcam), anti‐cleaved PARP1 (ab2302; Abcam), anti‐MPO (ab9535; Abcam), anti‐TLR4 (ab13556; Abcam), anti‐NF‐κB (BM3940, Boster), anti‐IL‐1β (ab23437; Abcam), anti‐IL‐18 (ab71495; Abcam), anti‐TNF‐α (ab1793; Abcam), anti‐C3 (ab200999; Abcam), and anti‐β‐actin (M01263‐2, Boster), followed by secondary antibodies conjugated to horseradish peroxidase anti‐rabbit IgG (H + L) (AS014, ABclonal) and anti‐mouse IgG (H + L) (AS003, ABclonal).
4
Comprehensive Western Blot Analysis Protocol
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Western blot analysis was performed as previously described [24 (link)]. Briefly, total protein was isolated from cells using the RIPA lysis buffer (Beyotime). After measuring the protein concentration using a BCA kit (Beyotime), a total of 40 μg protein was separated by 8% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto the polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). Membranes were subsequently blocked with 5% skim milk at room temperature for 2 h, and incubated at 4°C overnight with primary antibodies: anti-CCT3 (Abcam, Cambridge, UK, ab225878, 1:2,000), anti-cyclin B1 (Abcam, ab181593, 1:2,000), anti-cyclin dependent kinase 1 (CDK1, Abcam, ab133327, 1:10,000), anti-Cleaved Caspase-3 (Abcam, ab2302, 1:500), anti-STAT3 (Abcam, ab119352, 1:5,000), anti-p-STAT3 (Abcam, ab76315, 1:5,000), anti-JAK2 (Cell Signaling Technology, Inc., MA, USA, #3230, 1:1,000), anti-p-JAK2 (Cell Signaling Technology, #4406, 1:1,000), and anti-GAPDH (Beyotime, AG019, 1:1,000). After incubation with corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime, A0208, A0216, 1:5,000) at room temperature for 2 h, membranes were visualized under a ChemiDoc XRS+ system (Bio-Rad). The protein bands were quantified using the Image J software (National Institutes of Health, Bethesda, USA).
5
Comprehensive Apoptosis and Autophagy Assay
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Anti-VEGFA (ab1316), anti-caspase-3 (ab179517) antibodies, and anti-cleaved caspase-3 (ab2302) were purchased from Abcam (USA). Anti-p62 antibodies were purchased from Santacruz (CA). Anti-LC3B/MAP1LC3B (NB100-2220) and anti-Beclin1/ATG6 (NB500-249) antibodies were purchased from Novus Corporation. Anti-β-actin antibodies were purchased from Santacruz (CA). Annexin V-FITC Apoptosis Detection Kit (#4830-01-k) were purchased from R&D Systems (USA).
6
Protein Expression Analysis in Kidney and IMCD3 Cells
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Whole-cell protein was extracted from kidneys or IMCD3 cells using Radio immunoprecipitation assay (RIPA) buffer (Thermo Scientific, USA). An equal amount of proteins (20 µg) was loaded on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was then blocked with 5% skim milk and incubated overnight at 4 °C with anti-DJ-1 (1:1,000, 2134S, CST, USA), anti-Ki67 (1:1,000, ab16667, Abcam, USA), anti-PCNA (1:2,000, ab18197, Abcam, USA), anti-Bax (1:500, 2772S, CST, USA), anti-Bcl-2 (1:500, ab196495, Abcam, USA), anti-α-Smooth muscle actin (SMA, 1:1,000, ab5694, Abcam, USA), anti-fibronectin (FN1, 1:1,000, ab2413, Abcam, USA), anti-TGF-β1 (1:1,000, ab92486, Abcam, USA), anti-p53 (1:5,000, ab26, Abcam, USA), anti-Cytc (1:5,000, ab133504, Abcam, USA), anti-cleaved-caspase 3 (1:1,000, ab2302, Abcam, USA), and anti-cleaved-caspase 9 (1:1,000, ab2324, Abcam, USA) antibodies. Subsequently, the goat-anti-mouse Horseradish Peroxidase (HRP)-conjugated IgG antibody (1:5,000, Cat. No. ab205718, Abcam, USA) was used as a secondary antibody. Finally, bands were developed with an Enhanced Chemiluminescence Kit (Thermo Scientific, USA) and quantified by Quantity One imaging software (BioRad, USA) normalized to β-actin (internal control).
7
Cleaved Caspase-3 Immunohistochemistry
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IHC staining was performed using a standard streptavidin-peroxidase method. After deparaffinization and unmasking epitopes, kidney sections were incubated with primary antibody against cleaved caspase 3 (1:500, ab2302, Abcam, Cambridge, MA, USA) at 4 °C overnight, followed by incubation with the secondary antibody for 20 min at room temperature. Then, kidney sections were incubated with horseradish peroxidase (HRP)-conjugated streptavidin peroxidase (1:40,000, ab7403; Abcam) for 30 min. DAB was used as the chromogen to visualize the results. Finally, kidney sections were counterstained with hematoxylin.
8
Western Blot Analysis of Protein Expression
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Radio Immunoprecipitation Assay (RIPA) lysis buffer, provided by Sigma (St. Louis, MO, USA), was used to collect protein lysates. BCA method was applied to quantify the total protein concentration. Proteins were added to SDS polyacrylamide gel electroelectrometry (PAGE), and electrotransferred to polyvinylidene fluoride (PVDF) membrane after electrophoresis separation. Blocking buffer (5% bovine serum albumin, BSA) was used to block the membranes at room temperature. After blocking, primary antibodies were used to incubate the membranes overnight at 4°C. Primary antibodies (Abcam, Cambridge, MA, USA) were used at the following dilutions: anti-PCNA (1:1000, ab18197), anti-cleaved-caspase-3 (1:1000, ab2302), anti-caspase-3 (1:500, ab13847), anti-Twist (1:2000, ab175430), anti-MMP-2 (1:1000, ab37150), anti-MMP-9 (1:1000, ab73734), anti-phosphorylation-p38 (p-p38) (1:1000, ab4822), anti-p38 (1:1000, ab31828), anti-p-JNK (1:1000, ab124956), anti-JNK (1:1000, ab179461), anti-p-ERK (1:1000, ab65142), anti-ERK (1:10,000, ab32537), anti-GAPDH (1:500, ab8245). After washing the membranes Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 4 times, the secondary antibody was added and incubated at 37°C for 30 min. GAPDH served as the loading control. All experiments were repeated 3 times.
9
Immunohistochemical Analysis of Cardiac Cell Markers
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AMTs were fixed using 4% paraformaldehyde/PBS for 30 minutes and embedded in 13% polyacrylamide gel. 150 μm thick sections were made using a vibratory microtome and stained for cardiac troponin-T (TNNT2, MS-295-P, Thermo Scientific, 1:150 dilution), alpha sarcomeric actinin (α-Actinin, EA53, Sigma, 1:200 dilution), connexin-43 (GJA1, ab11369, Abcam, 1:100 dilution), active caspase-3 (ab2302, Abcam, 1:100 dilution), NKX2-5 (sc-8697, Santa Cruz, 1:50 dilution), and phospho histone H3 (P-histone3, ab32107, Abcam, 1:100 dilution) primary antibodies and Alexa Fluor secondary antibodies. Stained samples were scanned using a confocal microscope and used to generate 3D projection images of the tissue sections in ImageJ. Proliferation and apoptosis were assessed by calculating the percentage of total nuclei positive for phospho histone H3 and active caspase-3, respectively. Myotubes were identified as actinin positive cells containing 5 or more nuclei, since human CMs can contain up to 4 nuclei70 (link).
10
Western Blot Analysis of Neuronal Markers
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The neurons were lysed using RIPA buffer (#9806, Cell Signaling Technology, Danvers, MA, USA). Protein concentration was determined with a Bicinchoninic protein assay (BCA) kit (7780S, Cell Signaling Technology, Danvers, MA, USA). All the protein samples were then electrophoresed via 10% SDS-PAGE (P0012A Beyotime, China). Later, the samples were transferred to polyvinylidene fluoride membranes (PVDF; Millipore, Bedford, MA) which were blocked by 5% skimmed milk at room temperature for 60 min. The membranes were then incubated with the following primary antibodies overnight at 4ºC: anti-GAPDH antibody (rabbit, #5174, 1 : 1000, Cell Signaling Technology, USA), anti-NGF antibody (rabbit, ab221609, 1 : 1000, Abcam, UK), anti-C-caspase-3 antibody (rabbit, ab2302, 1 µg/ml, Abcam, UK), anti-CNTF antibody (rabbit, ab46172, 1 : 3000, Abcam, UK) and anti-BDNF antibody (rabbit, ab108319, 1 : 1000, Abcam, UK). The membranes were then incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-rabbit IgG H&L (HRP) (ab205718, 1 : 2000, Abcam, UK) for 1 h at room temperature and washed with tris-buffer saline tween (TBST) three times. GAPDH was employed as the normalization reference. An enhanced chemiluminescence (ECL) plus kit (K22030, Abbkine Scientific, China) was employed to measure the labelled proteins [36] .
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