After two washings with cold PBS, cells were lysed in ice-cold lysis buffer (SDS 1% EDTA 10 mM; Tris-HCl pH8,1 50 mM; protease inhibitors, Na3VO4 1 mM, NaF 100 × ) and extracts were sonicated. Protein extracts were separated by SDS-PAGE, transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) and revealed with a chemiluminescence kit (Millipore). Presented Western-Blot are representative of three independent experiments. Following antibodies were used: actin (MAB1501R, Millipore), β-tubulin (T0198, Sigma), BAX (A3533, Dako), BCL-xL (1018-1, Epitomics), cytochrome c (BD-Pharmingen San Diego, CA, USA), MCL-1 (sc-819, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), NOXA (ALX-804-408, Enzo Life Science, New York, NY, USA), PUMA (12450, Cell Signalling), p21 (2947, Cell Signalling), p53 (#554294, BD-Pharmigen). The above antibodies against BAX, BCL-xL and p53 were also used for immunoprecipitations, as those against BAX 6A7 (ab5714, Abcam, Cambridge, UK) and FLAG-tag (F1804, Sigma).
Ab5714
Manufactured by Abcam
Sourced in United States
Ab5714 is a primary antibody product from Abcam. It is a recombinant monoclonal antibody that targets a specific protein epitope. The antibody is purified and provided in a buffered solution.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence kit, by Merck Group (1 mentions)
Actin mab1501r, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Agilent Technologies (1 mentions)
Mcl 1 sc 819, by Santa Cruz Biotechnology (1 mentions)
Cytochrome c, by BD (1 mentions)
Bcl xl, by Abcam (1 mentions)
β tubulin, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Ab79351, by Abcam (1 mentions)
Ab107166, by Abcam (1 mentions)
Ab6142, by Abcam (1 mentions)
Ab97959, by Abcam (1 mentions)
Mab377, by Merck Group (1 mentions)
Sc 7382, by Santa Cruz Biotechnology (1 mentions)
Ab13506, by Abcam (1 mentions)
Ab25138, by Abcam (1 mentions)
Ab18528, by Abcam (1 mentions)
Mbs1499661, by MyBioSource (1 mentions)
Anti flag m2 f1804, by Merck Group (1 mentions)
6 protocols using ab5714
1
Western Blot Analysis of Apoptosis Regulators
2
Histological and Immunohistochemical Analysis of Ischemic Stroke in Rats
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All rats used for the evaluation of infarction volume with MRI were sacrificed 48 hours after MCAO for histological and immunohistochemical analyses. Hematoxylin and eosin (H&E) staining distinguished the peri-infarct region from the remaining area. TUNEL-positive cells were counted in the GV1001 and control (sham and saline) groups. Immunohistochemical staining was performed as previously described [7 (link)-9 (link)] using antibodies against phosphorylated Akt (pAkt; Ser473, 1:100, 9271, Cell Signaling Technology, Beverly, MA, USA), phospho-glycogen synthase kinase (pGSK-3β; 1:100, ab107166, Abcam, Cambridge, MA, USA), phosphorylated-extracellular signal-regulated kinase (pERK)1/2 (Thr202/Tyr204; 1:1,000, 9101, Cell Signaling Technology), B-cell lymphoma 2 (Bcl-2; 1:100, sc-7382, Santa Cruz Biotechnology, Dallas, TX, USA), Bcl-2 associated X (Bax; 1:100, ab5714, Abcam), nestin (1:200, ab6142, Abcam), neuronal nuclei (NeuN; 1:100, MAB377, Millipore, Bedford, MA, USA), doublecortin (DCX; 1:100, ab28941, Abcam), and SRY-box transcription factor 2 (SOX2; 1:50, ab79351, Abcam; 1:50, ab97959, Abcam). The detailed methods are described in the Supplementary methods .
3
Subcellular Localization of Key Proteins
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Flag-RECS1, MYC-RECS1, BAX, ERp57, LAMP-1/2, LGALS1/galectin-1, and LGALS3/galectin-3 proteins were visualized by immunofluorescence. The following antibodies were used: anti-Flag (F7425, Sigma-Aldrich), anti-Flag M2 (F1804, Sigma-Aldrich), anti-TBMIM1 (MBS1499661, MyBioSource), anti-ERp57 (ab13506, Abcam), anti–LAMP-1 (an24170, Abcam), LAMP-2 (ab18528, Abcam), BAX (ab5714, Abcam), anti-LGAL1/galectin-1 (ab25138, Abcam), and LGAL3/galectin-3 (556904, BD Biosciences). All antibodies were diluted 1:1000. We used a sensitive method based on a confined displacement analysis algorithm to calculate colocalization coefficients between Flag-RECS1 and ERp57 (ER), GM130 (Golgi apparatus), and LAMP-1/2 (endosomes/lysosomes) (63 (link)). The colocalization of images was performed as previously described (63 (link)).
4
Western Blot Analysis of Apoptosis Markers
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Cells were washed with PBS and lysed on ice in RIPA buffer (V900854, Sigma) containing a cocktail of protease inhibitors (P8340, Sigma) following the manufacturers protocols. Protein concentration was measured using the BCA assay. The protein fractions were suspended in loading buffer and denatured at 100°C for 5 min. Total proteins (20 µg/lane) were separated on 12% SDS-PAGE and transferred to PVDF membranes, which were blocked in 5% fat free milk in TBST buffer (0.1% Tween-20) for 2 h at room temperature. BRCA1 levels were analyzed using a mouse monoclonal anti-BRCA1 antibody (1:1,000; ab16780; Abcam); Bcl-2, Bax, pro-caspase-3, cleaved-caspase-3, and cytochrome c levels were detected using the following mouse or rabbit monoclonal antibodies at a dilution of 1:1,000: ab117115, ab5714, ab13586, ab32042, and ab13575, respectively (Abcam). The secondary antibodies used were goat anti-rabbit antibody (1:1,000; Sc2030; Santa Cruz Biotechnology) and rabbit anti-mouse antibody (1:1,000; Sc358917; Santa Cruz Biotechnology). GAPDH was used as the endogenous control to normalize the expression levels of the proteins of interest. GAPDH levels were detected using HRP-conjugated mouse anti-GAPDH monoclonal antibody (1:5,000; HRP-60004, Proteintech). The densitometry scan of the western blotting was performed by Image Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
5
Hypoxia-Induced Cardiomyocyte Protein Profiling
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Eight hours after hypoxia, protein was isolated from cardiomyocytes with standard Invitrogen protocols (Invitrogen, Carlsbad, CA, USA). Protein concentration quantitation was modified by Bradford assay (Bio‐Rad Laboratories, Hercules, CA, USA), protein was then separated by SDSPAGE with antibodies against Mst1 (ab51134; Abcam), p‐Mst1 (Thr183) (#110687; Sigma‐Aldrich), LC3A/B (#12741S, CST), P62 (ab91526; Abcam), Beclin1 (ab62472; Abcam), GAPDH (ab181602; Abcam), Caspase‐3(ab4051; Abcam), Cleaved caspase‐3 (ab2302; Abcam), Bax (ab5714; Abcam), Bcl‐2 (ab7973; Abcam). The blots were visualized with a chemiluminescene system (Amersham Bioscience, Buchinghamshire, UK). The signals were quantified by Image Pro Plus software (Media Cybernetics, MD Rockville, USA).
6
Protein Extraction and Western Blot Analysis
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Proteins were extracted using an ice-cold RIPA lysis buffer (P00138, Beyotime, Nanjing, China) with proteinase inhibitor (ST506, Beyotime, Nanjing, China). Equal amounts of proteins were measured using an BCA protein assay kit (A045-3, Jiancheng, Nanjing, China). Proteins were separated by electrophoresis and then electronically transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, USA) using a BioRad system (BioRad, Hercules, USA). The membrane was blocked in 5% skimmed milk at room temperature for 2 h and subsequently incubated overnight at 4°C with corresponding primary antibodies, rabbit anti-BCL2 (1:200), mouse anti-GRP78 (1:200), rabbit anti-LC3β (1:100, sc-28266, Santa Cruz Biotechnology, Santa Cruz, USA), rabbit anti-CYP11A1 (1:200, CSB-PA006389LA01HU, CusAb, Wuhan, China), mouse anti-BAX (1:100, ab5714) and mouse anti-β-actin (1:1000, ab8226, Abcam, Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (sc-2004 or sc-2005, Santa Cruz Biotechnology, Dallas, USA) were then used to detect proteins using Clarity ECL Western Blot Substrate kits (BioRad, Hercules, USA) and exposed using a ChemiScope 3400 Mini machine (Clinx, Shanghai, China). Protein quantification was performed using densitometry analyses on Quantity One Software.
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