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Bcl2 associated x (bax)

Manufactured by ABclonal
Sourced in China, United States, United Kingdom

Bax is a laboratory equipment product offered by ABclonal. It is a protein that plays a key role in the regulation of apoptosis, a process of programmed cell death. Bax functions as a pro-apoptotic member of the Bcl-2 protein family, which are involved in the control of mitochondrial outer membrane permeabilization. The product is designed for use in scientific research applications.

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54 protocols using bcl2 associated x (bax)

1

Western Blot Analysis of Cellular Proteins

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Total protein extraction from treated and untreated cells was performed using M-PER (including a protease inhibitor cocktail) followed by gel electrophoresis (SDS-PAGE) before being blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was incubated in the blocking buffer (5% skimmed milk in TBST buffer), then treated with primary antibodies, including PDX1 (1:3000, #ab47267, Abcam, Cambridge, UK), BAD (A19595), BAX (A2211), PARP (A19596), AKT (#A17909), BCL-2 (#A19693), Phospho-AKT-S473 (#AP0637), and PI3Kinase p110 (#A19742), and from Abclonal technology (Woburn, MA, USA), Pro/Insulin (#81385), from Cell Signaling Technology (CA, USA) or β-actin (#A5441, Sigma-Aldrich, Hamburg, Germany) antibodies. Finally, the membrane was treated (for one hour at room temperature) with the secondary antibodies (#7076S and #7074S, Cell Signaling Technology, Santa Cruz, CA, USA). Chemiluminescence was detected using a Bio-Rad Enhanced chemiluminescence (ECL) substrate kit (Bio-Rad, Hercules, CA, USA). Protein bands were detected using Bio-Rad Image Lab software (Bio-Rad, Hercules, CA, USA). Image J software was used to calculate the band counts. As an endogenous control in all studies, β-actin was employed.
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2

Protein Extraction and Western Blotting

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Total protein was extracted using RIPA lysate (Beyotime, Nantong, China) containing protease inhibitors and phosphatase inhibitors (CWBIO, Taizhou, China), and protein concentration was determined by the BCA protein assay kit (Beyotime, Nantong, China). Proteins were denatured in 5 × SDS-PAGE sample loading buffer (YEASEN, Shanghai, China), electrophoretically separated, and transferred onto PVDF membranes. After blocking in 5% skimmed milk for 2 h at room temperature, the proteins were incubated overnight at 4 °C with corresponding primary antibodies, including HSP90 (Proteintech, Wuhan, China), phospho-ERK1/2 (Cell Signaling Technology, Boston, MA, USA), ERK1/2 (Cell Signaling Technology, Boston, MA, USA), BCL2 (ABclonal, Wuhan, China), and BAX (ABclonal, Wuhan, China). Finally, the membranes were incubated in a horseradish peroxidase-labeled secondary antibody (CWBIO, Taizhou, China) for 2 h at room temperature. Protein expression was detected on a gel documentation system (Tanon, Shanghai, China) using ECL chemiluminescent chromogen solution (ABBKINE, Redlands, CA, USA).
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3

Western Blot Analysis of Cellular Proteins

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PC samples and cells were lysed in RIPA buffer supplemented by protease and phosphatase inhibitors (TargetMol) and incubated on ice for 30 minutes. The supernatant was collected after discarding the sedimentation. The denatured proteins were added to the chamber for SDS‐PAGE, followed by electrotransfer onto PVDF. After blocking with 3% of BSA for 1 hours, the membranes were incubated with diluted primary antibody overnight at 4°C. On the following day, the primary antibody was discarded and the membranes were washed three times with TBST, followed by incubation with diluted secondary antibody for 1 hours at room temperature. The immune complexes were detected via an enhanced chemiluminescence system (Life Tec). Analysis and quantification of the bands were performed using ImageJ software (Version 11). The primary antibodies involved in this work included the following: HELLS (1:1000; Abclonal), TGFBR3 (1:1000; Abclonal), γH2AX (1:1000; Abclonal), caspase 3/cleaved caspase 3 (1:1000; CST), caspase 9/cleaved caspase 9 (1:1000; CST), Bax (1:1000; Abclonal), and GAPDH (1:1000; Abclonal). Secondary antibodies were purchased from Abclonal, China.
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4

Western Blot Analysis of Apoptosis and Oxidative Stress

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Proteins were extracted from brain tissues of the ischemic side and PC12 cells, separated on 10%–15% SDS-PAGE gels, and transferred to membranes. Then, the membranes were incubated with primary antibodies against Bax (1 : 1000, ABclonal, China), Bcl-2 (1 : 1000, ABclonal, China), caspase-3 (1 : 1000, ABclonal, China), cleaved caspase-3 (1 : 1000, ABclonal, China), Nrf2 (1 : 1000, Cell Signaling Technology, USA), HO-1 (1 : 1000, Proteintech, USA), Lamin B (1 : 1000, Abcam, USA), GAPDH (1 : 1000, Proteintech, USA), and β-actin (1 : 1000, Proteintech, USA). After three washes with TBST, the membranes were incubated with corresponding secondary antibodies (1 : 6500, Abcam, USA) and scanned using a BIO-RAD imaging system (BIO-RAD Gel Doc XR, USA). The band intensity was normalized to the intensity of the GAPDH or Lamin B band and analyzed using Image Lab v5.2 software.
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5

Western Blot Analysis of Apoptosis Markers

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ESCC cells were plated into 6-well plates at a density of 2.5x105/well. After 24 hrs STA-9090 or vehicle was added into designated wells for 72h. The cells were lysed with RIPA Lysis Buffer and protein concentrations were measured with a BCA assay kit. The protein lysates were electrophoresed and transferred to PVDF membranes (GE Healthcare, Buckinghamshire, UK) and incubated overnight with the primary antibodies at 4°C. After washing with PBS the membranes were incubated with the appropriate special secondary antibodies at room temperature for 1h for signal detection. Antibodies used: HSP90 (1:1000) antibody and β-actin (1:10,000) antibody (Affinity Biosciences, OH, USA), BAX (1:500), BCL-2 (1:500), MYC (1:500) antibody (ABclonal Biotech Co, Woburn, MA, USA), Anti-mouse HRP (1:10,000) (ABclonal Biotech Co, Woburn, MA, USA), Anti-rabbit HRP (1:10,000) (ABclonal Biotech Co, Woburn, MA, USA).
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6

Western Blot Analysis of Apoptosis Markers

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CRC samples and cells were lysed in RIPA buffer supplemented by protease and phosphatase inhibitors (TargetMol, America) and incubated on ice for 30 min. The supernatant was collected after discarding the sedimentation. The denatured proteins were added to the chamber for electrophoresis in SDS-PAGE and subsequently transferred onto PVDF membranes by electrotransfer. After blocking with 3% BSA for 1 h, the membranes were incubated with diluted primary antibody overnight at 4 °C. On the following day, after discarding the primary antibody, the samples were washed three times with TBST and incubated with diluted secondary antibody for 1 h at room temperature. The immune complexes were detected via an enhanced chemiluminescence system (Life Tec, America). Analysis and quantification of the bands were performed by ImageJ software (Version 11). The primary antibodies involved in this work include KIF11 (1:1000, CST, America), γH2AX (1:1000, Abclonal, China), caspase 3/cleaved caspase 3 (1:1000, CST, America), caspase 9/cleaved caspase 9 (1:1000, CST, America), Bax (1:1000, Abclonal, China), Bad (1:1000, Abclonal, China), and GAPDH (1:1000, Abclonal, China). Secondary antibodies were purchased from Abclonal, China.
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7

Protein Extraction and Immunoblotting Analysis

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KUP5 and AML12 cells used for protein extraction were cultured in 6-well plates and treated with CDs concentrations of 0, 100 and 400 μg/mL. The upper medium was discarded and the cells were lysed as described previously [44 (link)]. The protein content was analyzed using the bicinchoninic acid (BCA) method and equal amounts (30 μg) of protein sample were loaded onto a 12.5% SDS-PAGE gel for separation, transferred to a PVDF membrane, and sealed with 5% skim milk powder diluted with TBST for 1 h. The samples were then incubated with primary antibody (1:1000) for Cleaved-caspase3, p62, Beclin1 (Cell Signal Technology (CST), USA), Bax, Bcl-2, GAPDH, TFEB, H3, p-mTOR, or p-ERK (ABclonal, Wuhan, China) at 4 °C overnight. Thermo32106ECL luminescence (Thermo Fisher Scientific, USA) measurements were performed after co-incubation with the corresponding secondary antibody (V: V = 1:10,000) at room temperature for 1 h. The Tanon MP imaging System (Tanon 5200, Shanghai, China) and ImageJ 1.53a were used for image acquisition and strip gray scale quantification.
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8

Protein Extraction and Western Blot Analysis

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Cells were lysed using the PRO-PREPTM Protein Extraction solution (iNtRon Biotech, Seongnam-Si, Republic of Korea). Total proteins were transferred to a polyvinylidene difluoride membrane and blocked with 5% skimmed milk for 1 h. Specific proteins were detected with primary antibodies: NLRP3 (A12694), GSDMD (A20197), IL-1β (A1112), caspase-3 (A19654), caspase-9 (A11451), BNIP3 (A5683), Cyt-c (A1561), Bax (A19684), Bcl-2 (A19693), β-actin (AC026), p-mTOR (S2448) (AP0115), mTOR (A2445), p-PI3K (AP0845), PI3K (A4992), p-Akt (AP0637), Akt (A17909), P62 (A19700), and LC3B (A19665), which were all bought from ABclonal Technology Co. Ltd., Wuhan, China. The blots were then treated with horseradish peroxidase-conjugated secondary antibodies. Bands were visualized using the ECL reagent and FluorChem M system (ProteinSimple, SFO, San Jose, CA, USA)
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9

Mitochondrial Dynamics and Apoptosis Signaling

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After lysing on ice for 30 min with RIPA containing 1% phenylmethanesulfonyl fluoride (PMSF, Beyotime, China), cells were centrifuged 12,000 g to collect supernatant (at 4 °C for 15 min). The supernatant was collected to quantify protein content by BCA kit (Thermo, USA). SDS-PAGE was used to separate the total protein, then transferred to an Immobilon™ PVDF membrane (Millipore, USA) was applied for 90 min at 220 mV. The blot was blocked with TBST (0.1% Tween-20 in Tris buffered saline) containing 5% skim milk powder for 2 h, and incubated with primary antibodies: Drp1, Fis1, Mfn1, Mfn2, OPA1, Caspase-3, Bax, Bcl-2 (their dilution ratio was all 1:1000, Abclonal, China), p-Drp1 S616 (1:1000, Affinity, USA) overnight at 4 °C. After washing three times with TBST, the PVDF membrane was incubated with horseradish peroxidase-coupled (HRP) Goat Anti-Rabbit IgG secondary antibody (1:5000, Abclonal, China) for 1 h. The image collection was performed using BeyoECL plus Western blotting detection system.
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10

Protein Expression Analysis in NSCLC

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Proteins in NSCLC samples, tumor bodies and cells were extracted and the protein concentration was determined with a bicinchoninic acid protein assay kit (Beyotime). Equal amount of proteins was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Thermo Fisher, Waltham, Massachusetts). Afterward, the membranes were blocked with 5% bovine serum albumin (Biosharp, Hefei, China) and then incubated at 4°C overnight with antibodies recognizing cyclinD (1:1000; Abclonal, Wuhan, China), cyclinE (1:1000; Proteintech, Wuhan, China), Bcl-2 (1:500; ABclonal), Bax (1:500; ABclonal), SOX4 (1:1000; Affinity, Changzhou, China) and β-actin (1:2000; Proteintech). After washing with tris-buffered saline with tween, the membranes were incubated at 37°C for 40 min with horseradish peroxidase-conjugated secondary antibodies (Proteintech). Finally, the membranes were visualized using a chemiluminescence detection system (7sea biotech, Shanghai, China). β-actin served as the internal control.
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