Proteins were separated and detected as previously described [28 (link)]. In brief, SDS-PAGE gel (Mini-Protean TGX Precast Gel 12%, 456–1045, Bio-Rad) was used to separate proteins and then transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Blocking of membranes was achieved by using 5% dry milk dissolved in Tris Buffer Saline with 1% Tween 20 (TBST) and the following antibodies were incubated overnight at 4 °C: rabbit anti-BCL-2 (CST4223, Cell Signaling), rabbit anti-BCL-xL (CST2764, Cell Signaling), rabbit anti-MCL-1 (CST5453, Cell Signaling), rabbit anti-NOXA (CST14766, Cell Signaling), rabbit anti-BIM (CST2933, Cell Signaling), rabbit anti-phospho-ERK1/2 (CST4376, Cell Signaling), rabbit anti-Actin (CST4970, Cell Signaling). Anti-rabbit IgG HRP-linked secondary antibody (CST7074, Cell Signaling) was used and immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad). When required, immunoblots were stripped in 0.1 M glycine pH 2,5, 2% SDS for 40 min and washed in TBS. The visualization of the bands was done using the LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ was then used to quantify the integrated optical density of bands.
Rabbit anti bcl xl
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-Bcl-xL is a primary antibody that recognizes the Bcl-xL protein, which is a member of the Bcl-2 family and plays a role in regulating apoptosis. This antibody is intended for use in various immunoassay applications to detect and study the Bcl-xL protein.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti bim, by Cell Signaling Technology (4 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (4 mentions)
Rabbit anti mcl 1, by Cell Signaling Technology (2 mentions)
Rabbit anti actin, by Cell Signaling Technology (2 mentions)
Rabbit anti bcl 2, by Cell Signaling Technology (2 mentions)
Rabbit anti bim, by BD (2 mentions)
Mouse anti bcl 2, by Agilent Technologies (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Zymo Research (2 mentions)
Luminata forte western hrp substrate, by Merck Group (2 mentions)
Rabbit anti gfp, by Cell Signaling Technology (2 mentions)
Rabbit anti mcl 1, by Santa Cruz Biotechnology (2 mentions)
Ez ecl chemiluminescence reagent, by Sartorius (2 mentions)
Biospectrum 500 imaging system, by Analytik Jena (2 mentions)
Mouse anti vinculin, by Merck Group (2 mentions)
Mouse anti bax, by BD (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Mouse anti noxa, by Enzo Life Sciences (2 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Mouse anti bcl 2, by Abcam (2 mentions)
Rabbit anti cleaved parp, by Cell Signaling Technology (2 mentions)
21 protocols using rabbit anti bcl xl
1
Quantitative Immunoblotting of Apoptosis Regulators
2
Antibody Reagent Identification for Cell Analysis
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The rabbit anti-Bcl-XL, the rabbit anti-caspase-3, the rabbit anti-LC3, the mouse anti-PARP1, the rabbit anti-actin antibodies were from Cell Signaling (ref. no. 2764, 9662, 2775, 9546, 4970 respectively). The rabbit anti-ATG6, the rabbit anti-total Bax and the rabbit anti-total Bak were from Santa Cruz Biotechnology (ref. no. sc-11427, sc-493 and sc-832 respectively). The mouse anti-caspase-1 was from Adipogen (ref. no. AG-20B-0048). The rabbit anti-ATG5 was from Abcam (ref. no. ab108327). The rat anti-MLKL was from Merck Millipore (ref. no. MABC604). The mouse FITC-labelled anti-CD19 was from Beckman Coulter (ref. no. A07768).
3
Apoptosis Regulators Antibodies Protocol
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ABT-263 was obtained from Selleck Chemicals (Houston, TX) and JC-1 from Cayman Chemicals (Ann Arbor, MI). Rabbit anti-Bcl-xL (#2764), Bcl-2 (#2870), Bim (#2819), PARP (#9532) and caspase-3 (#9665), and mouse anti-caspase-9 (#9808) were from the Cell Signaling Technology (Danvers, MA). Rabbit anti-Bak (sc-832), Bim (sc-11425), Puma (sc-28226) and Bcl-2 (sc-492), mouse anti-Puma (sc-374223), Bax (sc-70405, sc-23959), Bcl-2 (sc-509), Bcl-xL 9sc-8392), cytochrome c (sc-13560), and β-actin (sc-1616), and goat anti-Puma (sc-19187) were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-Bim (#559685), mouse anti-cytochrome c (#556433), and hamster anti-mouse Bcl-2 (#554218) were provided by BD Pharmingen. Rat anti-Bim (#804-528) was from Enzo Life Sciences (Farmingdale, NY) and Mouse ant-Bak (#06-536) was from EMD Millipore (Billerica, MA).
4
Analyzing Protein Expression in Drug-Treated Cells
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Adherent and non-adherent cells were collected from drug-treated dishes and total protein extracted by Tris-SDS lysis buffer, quantified by Lowry and used for immunoblotting. Antibodies used in the study were rabbit anti-Bcl-xL (1:1000, #2764; Cell Signaling), mouse anti-Bcl-2 (1:500, Dako; #M0887), mouse anti-PCNA (1:2000,#MS106-PO; Neomarker), rabbit anti-p89-PARP (1:1000, #9541; Cell Signaling) and rabbit-anti-Acetyl-H3 (1:1000, #06-599; Millipore). Equal protein loading between lanes was confirmed by staining membranes with Ponceau S prior to immunoblotting.
5
Immunoblotting Protein Detection Protocol
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For immunoblotting, cells were seeded in 24 or 96 well plates (Corning) then later harvested by trypsinisation, centrifuged, washed and lysed in SDS buffer (0.35 M Tris pH 6.8, 0.1 g/ml sodium dodecyl sulfate, 93 mg/ml dithiothreitol, 30% glycerol, 50 μg/ml bromophenol blue). Proteins were resolved by SDS-PAGE then electro-blotted onto Immobilion-P membranes (Millipore). Membranes were blocked in 5% dried milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) then incubated overnight at 4°C with the following primary antibodies diluted in milk: rabbit anti-Mcl-1 (Santa-Cruz), sheep anti-Tao1 [60 (link)], mouse anti-Cyclin B1 (Millipore), rabbit anti-Bcl-xL (Cell Signalling), sheep anti-Bub3 (Holland and Taylor, unpublished), rabbit anti-FBW7 (Bethyl), mouse anti-Bak (Calbiochem), rabbit anti-Bax (Santa-Cruz), mouse anti-myc tag (4A6, Millipore), rabbit anti-GFP (Cell Signalling). Following TBST washes, blots were incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies (Zymed). Bound secondaries were then detected by addition of EZ-ECL Chemiluminescence Reagent (Biological Industries) or Luminata™ Forte Western HRP Substrate (Millipore) and imaged using a Biospectrum 500 imaging system (UVP).
6
Western Blot Analysis of Apoptosis Regulators
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Protein extraction was done with TritonX-containing lysis buffer and protein content was determined using BCA assay (PierceTM BCA protein assay, Thermo Fisher Scientific). SDS–PAGE was carried out followed by semidry blotting. After blocking the membrane for 1 h with 5% milk, proteins were detected using the following antibodies: rabbit anti-MCL-1 (Enzo, ADI-AAP-240F), mouse anti-BCL-2 (Dako (Agilent), M088701-2), rabbit anti-BCL-xL (CellSignaling, 2762 S), mouse anti-NOXA (Enzo, ALX-804-408), mouse anti-BAX (BD Bioscience, 610983), rabbit anti-BAK (Upstate/ Merck, 06–536), rabbit anti-BIM (CellSignaling, 3183 S) and rabbit anti-caspase-3 (CellSignaling, 9662 S), mouse anti-β-Actin (Sigma, A5441), mouse anti-GAPDH (BioTrend (Hy Test Ltd), 5G4-6C5) or mouse anti-Vinculin (Sigma/Merck, V9131-100UL). Quantification of protein expression was performed using ImageJ 3.1 software.
7
Antibody Profiling for Apoptosis Signaling
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Antibodies used for immunoblotting analyses in this study are listed below: rabbit anti-MCL-1 (1:500, ab32087), rabbit anti-BAX (1:1000, ab32503) were obtained from Abcam; mouse anti-BCL-2 (1:1000, #15071), rabbit anti-BIM (1:1000, #2933), rabbit anti-BCL-XL (1:1000, #2762), rabbit anti-BAK (1:1000, #12105), rabbit anti-AKT (1:1000, #4691), rabbit anti-p-AKT (1:1000, #4060), rabbit anti-mTOR (1:1000, #2983), rabbit anti-p-mTOR (1:1000, #5536), rabbit anti-p-4EBP1 (1:1000, #2855), rabbit anti-4EBP1 (1:1500, #9644), rabbit anti-FOXO3a (1:1000, #12829), rabbit anti-p-FOXO3a (1:1000, #9466) rabbit anti-cleaved PARP (1:1000, #5625) and rabbit anti-cleaved Caspase-3 (1:1000, #9664) were purchased from Cell Signaling Technology; mouse anti-β-actin (1:1000, sc-47778), mouse anti-GAPDH (1:1000, sc-32233) and rabbit anti-PUMA (1:1000, sc-28226) were from Santa Cruz Biotechnology; rabbit anti-p-4EBP1 (1:500, NB100-81769, used in Fig. 1b ) and mouse anti-4EBP1 (1:1000, NBP1-47366, used in Fig. 1b ) were obtained from Novus Biologicals. The compounds of BH3 mimetics (ABT263 and ABT199) and mTOR inhibitors (BEZ235, AZD8055, and Temsirolimus) were from AbMole Bioscience.
8
Signaling Pathway Analysis in Cell Lines
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BCA (purity, 95.8%) was purchased from the Institute for Korea Traditional Medical Industry (Daegu, Korea) and dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). A BCA stock solution of 40 mM was stored at -80°C. Mouse anti-β-actin (1 : 5000 dilution), rabbit anti-p-AKT (1 : 1000 dilution), rabbit anti-AKT (1 : 1000 dilution), rabbit anti-p-p53 (Ser15) (1 : 1000 dilution), rabbit anti-p-p53 (Ser20) (1 : 1000 dilution), rabbit anti-p-p53 (Ser46) (1 : 1000 dilution), and rabbit anti-MDM2 (1 : 1000 dilution) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-p21 (1 : 1000 dilution), rabbit anti-p27 (1 : 1000 dilution), rabbit anti-p53 (1 : 1000 dilution), rabbit anti-FOXO3 (1 : 1000 dilution), rabbit anti-Bcl-2 (1 : 1000 dilution), rabbit anti-Bcl-xL (1 : 1000 dilution), rabbit anti-Bax (1 : 1000 dilution), rabbit anti-cleaved caspase-3 (1 : 1000 dilution), rabbit anti-caspase-3 (1 : 1000 dilution), rabbit anti-cleaved caspase-7 (1 : 1000 dilution), rabbit anti-caspase-7 (1 : 1000 dilution), rabbit anti-cleaved caspase-9 (1 : 1000 dilution), rabbit anti-caspase-9 (1 : 1000 dilution), rabbit anti-cleaved PARP (1 : 1000 dilution), and rabbit anti-PARP (1 : 1000 dilution) were purchased from Cell Signaling Technology (Danvers, MA, USA).
9
Immunoblotting Analysis of Cell Lysates
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Cell lysates were prepared in CHAPS-containing lysis buffer and analyzed by immunoblotting as described.28 (link) For detection of proteins by chemoluminescence (Advansta, Menlo Park, CA, USA; K-12049-D50), a rat anti-mA1 mAb, clone 6D6-1-1,52 (link) rat anti-Bim, clone 3C5 (Enzo, Lausen, Switzerland), rabbit anti-BclxL (Cell Signaling, Beverly, MA, USA; cs 2764 S), goat anti-Diva/Bcl2L10 (Santa Cruz, Dallas, TX, USA; sc8739), mouse anti-Bcl2 (BioLegend, London, UK; 633502), rabbit anti-Mcl1 (Rockland, Hamburg, Germany; 600-401-394) or a rabbit anti-GAPDH mAb (Cell Signaling, 2118, 1:5000) were used. Goat anti-rabbit Ig/HRP (Dako, Glostrup, Denmark; P0448), rabbit anti-goat Ig/HRP (Dako, P0449), goat anti-mouse heavy chain IgG1-HRP (Abcam, Cambridge, UK; ab98693) or rabbit anti rat-IgG heavy chain-HRP (Cell Signaling, 7077) were used as secondary reagents.
10
Western Blot Analysis of Apoptosis Regulators
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Western blot analysis was performed as previously described43 (link) using the following antibodies: mouse anti-NOXA, rat anti-BMF, rabbit anti-MCL-1 (Enzo Life Science, Farmingdale, NY, USA), mouse anti-BCL-2, rabbit anti-BAK (BD Biosciences), rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-BIM, mouse anti-PARP, rabbit-anti PUMA, rabbit-anti BCL-xL (Cell Signaling, Beverly, MA, USA), mouse anti-GAPDH (HyTest, Turku, Finland), rabbit anti-H3K4me2 (Diagenode, Liège, Belgium), rabbit anti-acetylated histone H3 (Merck Millipore, Darmstadt, Germany) and mouse anti-histone H3 (Abcam) or mouse anti-β-Actin (Sigma, Germany). Goat anti-mouse, goat anti-rabbit and goat anti-rat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and enhanced chemiluminescence (Amersham Biosciences, Freiburg, Germany) or infrared dye-labeled secondary antibodies and infrared imaging (Odysee Imaging System, LI-COR Biosciences, Bad Homburg, Germany) were used for detection. For detection of histone modifications cells were lysed using RIPA buffer supplemented with Pierce Nuclease (Thermo Fisher, Waltham, MA, USA). Representative blots of at least two independent experiments are shown.
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