Liver and kidney homogenates were prepared using 1× RIPA buffer with protease inhibitors. The protein concentration was measured using the Pierce BCA Protein Assay kit (Thermo Scientific). We used 30 ug of protein on an SDS-PAGE and then transferred it to either a nitrocellulose (Protran BA 83, Whatman) or a polyvinylidene difluoride (PVDF) membrane. We then blocked with 3% BSA-TBST (tris buffer saline with tween 20). The membranes were then probed with antibodies Cpt2 (Pierce), Pgc1α (Abcam), Acsl1 (Cell Signaling), Acot1 (Cell Signaling), Acot2 (Cell Signaling), Cpt1α (Abcam), Acadm (GeneTex), Acsf3 (Pierce), Total Acc (Cell Signaling), Acly (Cell Signaling), Hadha (Genetex), Aco2 (Cell Signaling), Fasn (BD Biosciences), and Hsc70 (Santa Cruz Biotechnology). Hsc70 used the appropriate Cy3 fluorescent secondary antibodies, and the other primary antibodies used the corresponding secondary antibodies conjugated to horseradish peroxidase. Images were collected using an Alpha Innotech FluorChemQ and presented with minimal image processing.
Protran ba83
Manufactured by Cytiva
Sourced in Germany
The Protran BA83 is a cellulose nitrate membrane filter designed for laboratory use. It has a pore size of 0.2 microns and a thickness of 0.16 millimeters. The membrane is composed of nitrocellulose.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Hsc70, by Santa Cruz Biotechnology (5 mentions)
Fluorchem q, by Bio-Techne (3 mentions)
Horseradish peroxidase labeled rabbit anti mouse, by Abcam (3 mentions)
Ecl detection system, by AbFrontier (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Supersignal west dura extended substrate, by Thermo Fisher Scientific (3 mentions)
Acadm, by GeneTex (2 mentions)
Total acc, by Cell Signaling Technology (2 mentions)
Hadha, by GeneTex (2 mentions)
Pgc 1α, by Abcam (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Ecl detection system, by Cytiva (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Phosstop, by Roche (2 mentions)
Complete mini, by Roche (2 mentions)
Western lightning ecl pro reagent, by PerkinElmer (2 mentions)
Mini protean gel, by Bio-Rad (2 mentions)
Enhanced chemiluminescence, by Bio-Rad (2 mentions)
35 protocols using protran ba83
1
Liver and Kidney Protein Expression
2
Comprehensive Western Blot Analysis of Cell Signaling
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Western blot analyses were performed as previously described [50 , 51 ]. Briefly, the cells were washed twice with PBS and lysed via sonication in lysis buffer (50 mM Tris-HCl, pH 7.5; 1% NP-40; 1 mM Na3VO4; 150 mM NaCl; 20 mM Na4P2O7; 100 mM NaF; 1% Na-deoxycholate; 0.1% SDS; 1 mM EDTA; phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Sigma)). The samples were separated on 6–15% SDS-PAGE gels, transferred to nitrocellulose membranes (Protran BA83, Whatman) and immunostained. The following primary antibodies and dilutions were used: Anti-p21 [C-19] 1:200 (Santa Cruz, #sc-397), anti-p53 [FL-393] 1:200 (Santa Cruz, #sc-6243), Anti-p16 (M-156) 1:200 (Santa Cruz, #sc-1207), anti-pRb 1:500 (BD-Pharmingen), anti-phospho-pRb (Ser807/811) 1:1000 (Cell Signaling), anti-phospho-histone H2A.X (Ser139) 1:1000 (Millipore 05–636) and monoclonal anti-α-tubulin 1:1000 (Sigma 9026). Horseradish peroxidase-labeled rabbit anti-mouse (Amersham, diluted 1:3000) and goat anti-rabbit (Abcam, #6721, diluted 1:3000) secondary antibodies were used. The proteins were visualized using an ECL detection system (Amersham Biosciences).
3
Western Blot Analysis of Adipose Tissue
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BAT, iWAT, and gWAT depots were homogenized with 300–500 ul of RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.25% deoxycholate) containing protease inhibitor cocktail (Roche) and phospho-STOP cocktail (Roche), followed by pelleting of the insoluble debris at 13,000×g for 15 min at 4 °C. The protein concentrations of lysates were determined by BCA assay (Thermo Scientific), and 30 μg of lysate was separated by Tris-glycine SDS-PAGE (10% polyacrylamide). Proteins were transferred to nitrocellulose membranes (Protran BA 83, Whatman), blocked in 3% BSA in 1X TBST (Tris-buffered saline with Tween 20), and incubated with primary antibodies overnight. The blots were probed with the following antibodies: Ucp1 (Sigma; U6382), Ndufb8, Sdhb, Uqcrc2, Atp5a (MitoProfile total OXPHOS, Abcam; ab110413), Aco2 (Cell Signaling; 6922), Mcad (GeneTex; GTX32421), Pcx (Abcam; ab128952), Vdac (Calbiochem; PC548), Pdh E2/E3bp (Abcam; ab110333), Acot2 (Sigma; SAB2100030), beta-Actin (Sigma; A2228), and Hsc-70 (Santa Cruz; sc-7298). Cy3-conjugated anti-mouse (Invitrogen), or HRP-conjugated anti-mouse (GE Healthcare) or anti-rabbit (GE Healthcare) secondary antibodies were used appropriately. Images were collected and analyzed using an Alpha Innotech FluorChemQ.
4
Drosophila Protein Extraction and Immunoblotting
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For each genotype, 15 brains from third-instar larvae were dissected in PBS, 1% protease inhibitor cocktail and extracted in 100 μl of cold protein extract buffer (50 mM trisHCl pH 7.4 (200923A, Euromedex), 50 mM NaCl (27810, Prolabo), 1 mM EDTA (E9884, Sigma), 0.25% DOC (D6750, Sigma), 0.1% SDS (EU0660, Euromedex), 1 mM PMSF (P7626, Sigma), 1‰, DTT (D0632), 1% of protease inhibitor solution (1 mM benzamidine-HCl (B6506, Sigma), 0,1 mg ml−1 phenanthroline (131377, Sigma), 1 mg ml−1 aprotinin (A6103, Sigma), 1 mg ml−1 leupeptin (L2884, Sigma), 1 mg ml−1 pepstatin A (P5318, Sigma)). Protein extracts were separated by SDS–polyacrylamide gel electrophoresis with either a 10% BIS-TRIS or a 3-8% TRIS-Glycine NuPAGE gel (Invitrogen) and then transferred on a nitrocellulose membrane (Protran BA83, Whatman). Membranes were probed with antibodies anti-Fzy (1:1,000, a gift from J. Raff lab) and anti-α-tubulin (DM1Α) (1:1,000, Sigma-Aldrich).
5
Western Blot Analysis of Apoptosis Markers
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Western blot analyses were performed as previously described [33 (link)]. Briefly, cells were washed twice with PBS and then lysed via sonication in lysis buffer (Intron, Seoul, Korea). The samples were separated on 10–15% SDS-PAGE gels, and then transferred to nitrocellulose membranes (Protran BA83; Whatman). Immunoblotting was performed using the following primary antibodies and dilutions: anti-Capase 8 (1:1000; Cell Signaling, Danvers, MA, USA), anti-Caspase 3 (1:1000; Cell Signaling, Danvers, MA, USA), anti-Caspase 7 (1:1000; Cell Signaling, Danvers, MA, USA), anti-cleaved Caspase 7 (1:1000; Cell Signaling, Danvers, MA, USA), PARP (1:1000; Cell Signaling, Danvers, MA, USA), BCL2 (1:1000; Cell Signaling, Danvers, MA, USA), DR4 (1:1000; Abcam, Cambridge, MA), DR5 (1:000; Abcam, Cambridge, MA, USA) and anti-beta actin (1:10000; Sigma, St. Louis, MO, USA). Horseradish peroxidase-labeled rabbit anti-mouse (Abcam, diluted 1:5000, Cambridge, MA, USA) and goat anti-rabbit (Santa Cruz, diluted 1:2000, Finnell Street, Dallas, TX, USA) secondary antibodies were used. The proteins were visualized using an ECL detection system (Ab Frontier, Seoul, Korea).
6
Western Blot Analysis of Signaling Proteins
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Western blot analyses were performed as previously described [18] (link), [19] (link). Briefly, cells were washed twice with PBS and then lysed via sonication in lysis buffer (Intron, Seoul, Korea). The samples were separated on 6%-15% SDS-PAGE gels and then transferred to nitrocellulose membranes (Protran BA83; Whatman). Immunoblotting was performed using the following primary antibodies and dilutions: anti–p-Akt (1:2000; Cell Signaling), anti–p-Akt (phospho Ser432) (1:1000; Cell Signaling), anti-mTor (1:2000; Cell Signaling), anti–p-mTor (1:1000; Cell Signaling), and anti–beta-actin (1:10000; Sigma, St. Louis, MO). Horseradish peroxidase–labeled rabbit anti-mouse (Abcam, diluted 1:5000) and goat anti-rabbit (Santa Cruz, diluted 1:2000) secondary antibodies were used. The proteins were visualized using an ECL detection system (Ab Frontier, Seoul, Korea).
7
Western Blot Analysis of E. histolytica Proteins
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Total protein extracts were prepared by lysing E.histolytica trophozoites (6×106/ml) with 1% Igepal (Sigma) in PBS using a previously described protocol [12] (link). Proteins (40 µg) in the extract were resolved on a 12% SDS-polyacrylamide gel in SDS-PAGE Running Buffer (25 mM Tris, 192 mM glycine, 0.1% SDS), and then electrotransferred in protein transfer buffer ((25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) to nitrocellulose membranes (Whatman, Protran BA83). The blots were first blocked using 3% skim milk, and then probed with 1∶500 rabbit polyclonal enolase antibody (Sc-15343; Santa Cruz Biotechnology), 1∶1000 monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (MAB374; Millipore), or 1∶500 rabbit polyclonal Gal/GalNAc lectin antibody (a kind gift from of N. Guillen, Pasteur Institute, Paris, France) for 16 hours at 4°C. After incubation with the first antibody, the blots were incubated with 1∶5000 secondary antibody for one hour at room temperature (Jackson ImmunoResearch), and then developed by enhanced chemiluminescence.
8
Western Blot Analysis of Metabolic Proteins
9
Western Blot Analysis of Aurora-A Kinase
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Mitotic cells collected by shake off were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 1 mM EGTA, 0.25% sodium deoxycholate) supplemented with protease and phosphatase inhibitors. Proteins were resolved by electrophoresis on 10% Laemmli gel and transferred on a nitrocellulose membrane (Protran BA83, Whatman) using a semi-dry system (BIO-RAD). 40 μg of extract per lane were loaded. Blocking and antibody incubations were performed at room temperature in PBS/0.1% Tween 20/5% low fat milk, or in PBS/0.1% Tween 20/5% BSA (for anti-phospho-Aurora-A hybridization). Antibodies were: mouse anti-Aurora-A (1 μg/ml; BD Transduction Laboratories), rabbit anti-phospho-Aurora-A (Thr288) (1:1000; C39D8; Cell Signaling Technology), goat anti-actin (0.5 μg/ml, I-19; SantaCruz Biotechnology). Signals were visualized by enhanced chemiluminescence detection (ECL plus, GE Healthcare, and Protein Detection System, GeneSpin).
10
Western Blot Analysis of TES-Ag Proteins
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TES-Ag proteins were electrophoretically separated using Criterion TGX (Bio-Rad, Cat. # 567–1092) at 10 ng/mm and then transferred to nitrocellulose membrane (Whatman Protran BA83, Cat. # 10 541 103, 0.2m pore size).The blots were cut into 2.5 mm strips and stored in PBS + 0.1% NaN3 at 4°C prior to use. Sera were tested and specific antibodies were detected as described previously [29 (link), 30 (link)]. A serum was considered positive if reactivity occurred with any bands at 24, 28, 30, or 35 kDa [31 (link), 32 (link)].
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