Protein phosphatase inhibitor and protease inhibitor cocktails were purchased from Sigma-Aldrich. VCP inhibitor Eer I and proteasome inhibitor MG132 were from Tocris Bioscience. Antibodies for Tom20 (sc-11415, 1:1,000), c-Myc (sc-40, 1:1,000), GFP (sc-9996, 1:1,000), GST (sc-138, 1:500), CD3 (sc-20047, 1:500), Enolase (sc-15343, 1:1,000), Tim23 (sc-514463, 1:500) and Parkin (sc-32282, 1:1,000) were from Santa Cruz Biotechnology. Full-length Htt (MAB2166, 1:1,000), polyQ (MAB1574, 1:1,000), EM48 (MAB5374, 1:1,000) and NeuN (MAB377, 1:500) antibodies were from Millipore. Pan-actin (A1978, 1:10,000) and Flag (F3165, 1:5,000) antibodies were from Sigma-Aldrich. Antibodies for VDAC (ab14734, 1:2,000), Clpp (ab124822, 1:1,000), UBXD1 (ab103651, 1:500) and VCP (ab109240, 1:10,000) were from Abcam. EEA1 (3288, 1:500) and LC3 (2775, 1:1,000) antibodies were from Cell Signalling, WFS1 (NB100-1918, 1:1,000) antibody was from Novus, HMGB1 (10829-1-AP, 1:1,000) antibody was from Proteintech, and GRP78 (ADI-SPA-826, 1:1,000) and Calnexin (ADI-SPA-860, 1:1,000) antibody was from Enzo Life Sciences. Anti-mouse IgG and anti-rabbit IgG, peroxidase-linked, species-specific antibodies were from Thermo Scientific.
Enolase
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Enolase is an enzyme that catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate, an essential step in the glycolytic pathway. It is widely used in various biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Tom20, by Santa Cruz Biotechnology (2 mentions)
Flag f3165, by Merck Group (1 mentions)
Tim23, by Santa Cruz Biotechnology (1 mentions)
Mg132, by Bio-Techne (1 mentions)
Parkin, by Santa Cruz Biotechnology (1 mentions)
Full length htt mab2166, by Merck Group (1 mentions)
Pan actin a1978, by Merck Group (1 mentions)
Hmgb1 10829 1 ap, by Proteintech (1 mentions)
Calnexin adi spa 860, by Enzo Life Sciences (1 mentions)
Ab14734, by Abcam (1 mentions)
Protein phosphatase inhibitor, by Merck Group (1 mentions)
Ab124822, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Electrogenerated chemiluminescence, by GE Healthcare (1 mentions)
β actin, by Abcam (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
V atpase b1 2, by Santa Cruz Biotechnology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
6 protocols using enolase
1
Antibody Specifications for Protein Analysis
2
Mitochondrial Protein Expression Analysis
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Samples were run on a 4–12% gel and transferred onto a nitrocellulose membrane. Membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween-20 and subsequently incubated with primary antibodies against GAPDH (1∶1,000; Santa Cruz), TOM20 (1∶1,000; Santa Cruz), F1F0-ATPase α subunit (1∶50,000; Abcam, Cambridge, MA), neuronal NO synthase (1∶1,000; Santa Cruz), endothelial NO synthase (1∶1,000; Santa Cruz), β-actin (1∶1,000, Abcam), or enolase (1∶1,000, Santa Cruz) overnight at 4°C. Membranes were then probed with the corresponding secondary antibodies for 1 hour and visualized by electrogenerated chemiluminescence (GE Healthcare, Piscataway, NJ). Densitometry was assessed using ImageJ software (NIH, Bethesda, MD).
3
Mitochondrial Bax Protein Estimation
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In order to estimate the mitochondrial Bax protein, the mitochondrial fraction was prepared using a test kit (Pierce, cat#89874) according to the manufacturer's instructions. The purity of the protein fraction was confirmed by immunoblotting with enolase (Santa Cruz Biotechnology) and VDAC (Thermo Fisher Scientific) antibodies for cytosol and for mitochondria, respectively.
4
Mitochondrial Protein Expression Analysis
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Protein concentrations of mitochondrial fractions harvested from mouse brains were determined by Bradford assay. Thirty μg of proteins were resuspended in Laemmli buffer, loaded on SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were probed with the indicated antibodies followed by visualization by ECL, and were then quantitated using NIH ImageJ software. The antibodies used in this study include Drp1 (1:2000, B&D Bioscience), p53 (1:500, Santa Cruz Biotechnology), Bax (1:1000, Proteintech Group Inc), PUMA (1:200, Proteintech Group Inc), voltage-dependent anion channel (VDAC, 1:2000, Abcam), Tom20 (1:5000, Santa Cruz Biotechnology) and Enolase (1:1000, Santa Cruz Biotechnology).
5
Western Blot Analysis of Macrophage Proteins
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BMDMs were lysed in RIPA buffer supplemented with 1X protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein was estimated using Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Equal amount of proteins was separated in 10% or 12% SDS-PAGE gels.
Proteins were then transferred onto a PVDF membrane and probed with antibodies against Glut1 (sc-7903, Santa Cruz Biotechnology), Enolase (sc-31859, Santa Cruz Biotechnology), HKX-2 (sc-6521, Santa Cruz Biotechnology), v-ATPase A1 (V0 subunit, sc-28801, Santa Cruz Biotechnology), v-ATPase b1/2 (V1 subunit, sc-21209, Santa Cruz Biotechnology), H2-I/Abβ (sc-71201, Santa Cruz Biotechnology), phospho-p38 (#9216, Cell Signaling), p38 (#9212, Cell Signaling), phospho-p65 (#3033, Cell Signaling), p65 (#4764, Cell Signaling), HIF1α (NB100-105, Abcam), MondoA (SAB2104303, Sigma-Aldrich), calnexin (sc-11397, Santa Cruz Biotechnology) or β-actin (sc-47778, Santa Cruz Biotechnology). Either calnexin or βactin was used as loading control. After incubation with secondary HRP-conjugated antibody (R&D) blots were developed using ECL reagent (GE Healthcare).
Proteins were then transferred onto a PVDF membrane and probed with antibodies against Glut1 (sc-7903, Santa Cruz Biotechnology), Enolase (sc-31859, Santa Cruz Biotechnology), HKX-2 (sc-6521, Santa Cruz Biotechnology), v-ATPase A1 (V0 subunit, sc-28801, Santa Cruz Biotechnology), v-ATPase b1/2 (V1 subunit, sc-21209, Santa Cruz Biotechnology), H2-I/Abβ (sc-71201, Santa Cruz Biotechnology), phospho-p38 (#9216, Cell Signaling), p38 (#9212, Cell Signaling), phospho-p65 (#3033, Cell Signaling), p65 (#4764, Cell Signaling), HIF1α (NB100-105, Abcam), MondoA (SAB2104303, Sigma-Aldrich), calnexin (sc-11397, Santa Cruz Biotechnology) or β-actin (sc-47778, Santa Cruz Biotechnology). Either calnexin or βactin was used as loading control. After incubation with secondary HRP-conjugated antibody (R&D) blots were developed using ECL reagent (GE Healthcare).
6
TPCN1 Protein Expression Analysis
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The protein extracted from TPCN1 KO and WT mice left ventricle was subjected to 2-DE Western blot analysis. 100 μg of protein was loaded onto pH 4-7 (heart-type fatty acid-binding protein (HFABP) and enolase) or 3-10 (phosphoglycerate kinase 1 (PGK1)) 7 cm IPG strips (GE Healthcare, UK). 2-DE was performed on 12% SDSpolyacrylamide gels. At the same time, we performed 1-DE SDS-PAGE/Western blot as previously described (González-Juanatey et al. 2003) . We used antibodies against HFABP (Santa Cruz Biotechnology, USA), enolase (Santa Cruz Biotechnology, USA) and PGK1 (R&D systems, USA) according to manufacturer instructions. The membranes were subsequently incubated with a horseradishperoxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA) and subjected to chemiluminescence detection (Millipore Corporate, USA). In all cases, densitometric analyses were performed using the UVP EC3 Imaging System (Ultra-Violet Products Ltd) and the Image J program (V1.43 q; Rasband 1997-2008).
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