Each of the WT-APP and the 3xK-APP expressing cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer (Tris-HCl, pH 7.4 50 mM, NaCl 150 mM, Triton X100 1%, Sodium deoxycholate 0.5%, SDS 0.1%) [36] (link). For APP dimer/monomer detection, we utilized 3–8% Tris-Acetate gels (Biorad) and for CTF detection we used 10% Bis-Tris gels (Biorad). The lysates were subsequently used for immunoblotting and detection of full-length APP and carboxyl terminal fragments (CTFs). APP C-terminal specific polyclonal antibody A8717 (1∶500) (Sigma-Aldrich) was used for the APP and CTFα/β detection. For recombinant substrate detection, we used 4–12% Bis-Tris gels (Biorad) and detected with monoclonal 6E10 antibody (1∶1000) (Covance, Emeryville, CA, USA). The blots were developed using an Odyssey infrared scanner (LiCor Biosciences, Lincoln, NE, USA).
Bis tris gel
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Bis-Tris gels are polyacrylamide gel electrophoresis (PAGE) products used for the separation and analysis of proteins. They are designed to provide high-resolution protein separation under neutral pH conditions.
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Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (14 mentions)
Odyssey infrared imaging system, by LI COR (9 mentions)
Ripa buffer, by Thermo Fisher Scientific (9 mentions)
Bca kit, by Thermo Fisher Scientific (9 mentions)
Tris acetate gel, by Bio-Rad (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Clarity western ecl substrate, by Bio-Rad (6 mentions)
β actin, by Cell Signaling Technology (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Irdye 800cw, by LI COR (5 mentions)
Image reader las 4000, by Fujifilm (5 mentions)
Bca assay, by Thermo Fisher Scientific (5 mentions)
Irdye 800cw labeled secondary antibody, by LI COR (4 mentions)
Image studio, by LI COR (4 mentions)
Image lab software, by Bio-Rad (4 mentions)
Iblot transfer system, by Thermo Fisher Scientific (3 mentions)
Stripping buffer, by Thermo Fisher Scientific (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Peroxidase labeled anti rabbit igg, by Vector Laboratories (3 mentions)
Peroxidase labeled anti mouse igg, by Vector Laboratories (3 mentions)
123 protocols using bis tris gel
1
Amyloid Precursor Protein Dimer Detection
2
Muscle Protein Quantification Procedure
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Whole EDL and soleus muscles were weighed and homogenised in ice-cold lysis buffer as described previously [37 (link)] and protein concentration determined by bicinchoninic acid protein assay (Pierce Biotechnology (Rockford, USA)). For measuring the abundance of PPARα, oxidative phosphorylation (OXPHOS) complexes I-V, peroxisome proliferative activated receptor γ coactivator 1α (PGC1α), CPT1b and β-tubulin proteins, 16–20 μg of muscle protein was subjected to SDS-PAGE using precast 10% Bis-Tris gels or 4–12% Bis-Tris gels (Bio-Rad Laboratories Pty Ltd (Gladesville, Australia)) and transferred to PVDF membranes. Membranes were incubated overnight in primary antibody; PPARα (Abcam (Cambridge, UK)), 1:500; OXPHOS proteins (MitoSciences (Eugene, USA)), 1:250; PGC1α (Abcam), 1:1000; CPT1b (Alpha Diagnostic International Inc (San Antonio, USA)), 1:1000; and β-tubulin (Cell Signaling Technology (Danvers, USA)), 1:2000. Bound primary antibodies were detected with sheep anti-rabbit (1:2500, Chemicon International (Billerica, USA)) or goat anti-mouse alkaline-phosphatase linked antibody (1:2000, Millipore (Billerica, USA)). Membranes were developed with chemifluoresence substrate (ECF), scanned by Typhoon Imager (GE Healthcare Bio-Sciences (Rydalmere, Australia)) and were quantified using ImageQuant software (Molecular Dynamics).
3
Immunoblot Analysis of Protein Extracts
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Immunoblot analyses were performed as previously described15 , 16 . Briefly, proteins were extracted in enzyme immunoassay buffer containing 250 mmol/L Tris base, 750 mmol/L NaCl, 5% NP‐40, 25 mmol/L EDTA, 2.5% sodium deoxycholate, 0.5% sodium dodecyl sulfate, and an EDTA‐free protease and phosphatase inhibitors cocktail tablet (Roche Applied Science, Indianapolis, IN, USA), sonicated, centrifuged at 56,700 g for 45 min at 4°C, and supernatants used for immunoblot analysis, as previously described. Total protein concentration was determined by using BCA Protein Assay Kit (Pierce, Rockford, IL). Samples were electrophoretically separated using 10% Bis–Tris gels or 3–8% Tris–acetate gel (Bio‐Rad, Richmond, CA), according to the molecular weight of the target molecule, and then transferred onto nitrocellulose membranes (Bio‐Rad). They were blocked with Odyssey blocking buffer for 1 h; and then incubated with primary antibodies overnight at 4°C. After three washing cycles with T‐TBS, membranes were incubated with IRDye 800CW or IRDye 680CW‐labeled secondary antibodies (LI‐COR Bioscience, NE) at 22°C for 1 h. Signals were developed with Odyssey Infrared Imaging Systems (LI‐COR Bioscience). Primary antibodies used in this paper are summarized in Table 1 . Actin was always used as an internal loading control.
4
Western Blot Analysis of Brain Cortex Proteins
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RIPA fractions of brain cortex homogenates were used for Western blot analyses as previously described (Di Meco et al., 2014 ; Chu et al., 2015 ). Briefly, samples were electrophoresed on 10% Bis–Tris gels or 3–8% Tris–acetate gel (Bio‐Rad, Richmond, CA, USA), transferred onto nitrocellulose membranes (Bio‐Rad), and then incubated overnight with the appropriate primary antibodies. Antibodies against 5LO, 12/15LO, DNMT1, DNMT3α, and DNMT3β were obtained from Santa Cruz Biotech. (Dallas, TX, USA) and SAH hydrolase (AHCY) antibody from Novus Biologicals (Littleton, CO, USA). After three washings with T‐TBS, membranes were incubated with IRDye 800CW‐labeled secondary antibodies (LI‐COR Bioscience, Lincoln, NE, USA) at room temperature for 1 h. Signals were developed with Odyssey Infrared Imaging Systems (LI‐COR Bioscience). β‐Actin was used as internal loading control.
5
Tau Oligomer Detection Protocol
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For the detection of tau oligomers, cortices were homogenized in PBS with protease and phosphatase inhibitors cocktail tablet (Roche Applied Science, Indianapolis, IN, USA, using a 1:3 dilution of tissue: PBS (w/v). Samples were centrifuged at 9,600 g for 10 min at 4°C, aliquoted and stored at −80°C until use (Lasagna‐Reeves et al., 2012 ). PBS‐soluble fractions were run on a 10% Bis–Tris gels (Bio‐Rad, Richmond, CA, USA), subsequently transferred onto nitrocellulose, blocked 1h at room temperature, and were probed overnight at 4°C with anti‐tau oligomer antibody T22 (1:1,000).
6
Western Blot Analysis of Brain Proteins
7
Detecting PfEMP1 in Enriched Infected RBCs
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Infected RBCs were enriched by gelatin flotation and incubated for 15 min with either 0, 10 or 100 μg ml−1 of trypsin (Baruch et al., 1996 (link)). Following three washes in PBS containing protease inhibitors, cells were either incubated with pooled hyperimmune serum (1:5) or proteins extracted with 1% Triton X-100 (v/v) followed by 2% SDS. Extracts were resuspended in sample buffer, heated, separated on 10% Bis-Tris gels (BioRad), and transferred onto nitrocellulose membranes (iBlot, Life Technologies). Detection of PfEMP1 was performed by incubation with a guinea pig anti-ATS antibody (1:400) followed by anti-guinea pig alkaline phosphatase (AP)-conjugated secondary antibody. Equal protein loading was verified with the mAb1C11 anti-Hsp70 antibody (1:2000) and revealed with NBT/BCIP following incubation with an anti-mouse AP-conjugated secondary antibody.
8
Protein Extraction and Western Blot Analysis
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Cells were lysed in SDS lysis buffer (0.1 M TRIS pH 7.5, 10% SDS, 1 mM NaVO3, and Complete protease inhibitor mix (Roche)) heated to 90 °C. Protein concentrations were determined using DC assay (BioRad, Hercules, CA). Samples were mixed with sample loading buffer (Life Technologies). Samples and BenchMark ladder (Life Technologies) were separated in BioRad Tris/Glycine/SDS running buffer on pre-made 10% Bis-Tris gels (BioRad). Proteins were transferred to PVDF membranes using Trans-Blot Turbo transfer system (BioRad), Ponceau S stained, and blocked for 1 h at 37 °C in 5% non-fat dry milk in TBST (0.01 M Tris/HCl, 0.15 M NaCl, 0.1% Tween 20, pH 7.4). Densitometric analyses were carried out using UN-SCAN-IT 6.1 software (Silk Scientific, Orem, Utah).
9
Western Blot Analysis of Mouse Brain
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RIPA (radio immunoassay precipitation) extracts from mouse brain homogenates were used for Western blot analyses as previously described (Di Meco et al., 2014 ; Giannopoulos et al., 2013 ). Briefly, samples were electrophoresed on 10% Bis–Tris gels or 3%–8% Tris–acetate gel (Bio‐Rad, Richmond, CA, USA), transferred onto nitrocellulose membranes (Bio‐Rad), and then incubated overnight at 4°C with the appropriate primary antibodies; anti‐5LO [dilution: 1:200] (Santa Cruz, Dallas, TX, USA), anti‐HT7 [1:200] (Thermo, Waltham, MA, USA), anti‐AT8 [1:100] (Thermo), anti‐AT180 [1:200] (Thermo); anti‐AT270 [1:200] (Thermo), anti‐PHF1 (generous gift of Dr. Peter Davies); anti‐PHF13 [1:100 (Thermo)], anti‐SYP [1:300] (Santa Cruz), anti‐PSD95 [1:200] (Thermo), anti‐MAP2 [1:1,000] (Millipore), anti‐GSK3α/β [1:100] (Cell Signaling, Danvers, MA, USA), anti‐pGSK3α/β [1:100] (Cell Signaling), anti‐cdk5 [1:200] (Santa Cruz), anti‐p35/p25 [1:100] (Santa Cruz), anti‐GFAP (Santa Cruz), anti‐CD45 [1:100] (Thermo) and anti‐Beta actin [1:500] (Santa Cruz). After three washings with T‐TBS (pH7.4), membranes were incubated with IRDye 800CW‐labeled secondary antibodies (LI‐COR Bioscience, Lincoln, NE, USA) at room temperature for 1 hr. Signals were developed with Odyssey Infrared Imaging Systems (LI‐COR Bioscience). β‐Actin was always used as internal loading control.
10
SUMO1 Immunoprecipitation and Proteomic Analysis
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Whole brain samples from Non-Tg or SUMO1+/+ Tg mouse were homogenized in lysis buffer (50 mM HEPES-KOH pH 8.0, 100 mM KCl, 2mM EDTA, 0.5% NP-40, 10% glycerol and protease inhibitors). Lysates were next incubated for 30 minutes at 4 °C, samples were centrifuged at high speed (15,000 g) and supernatants were collected. Protein lysates were first subjected to pre clearing with protein G-Sepharose 4 fast flow (GE Healthcare). SUMO1 polyclonal antibody (400 μg total) or preimmune rabbit serum conjugated beads were added to 120 mg of non-Tg or SUMO Tg mouse total brain lysates. Immunoprecipitated proteins were elutated with in Laemmli buffer and resolved by SDS-PAGE (4–12% Bis-Tris gels, BioRad) before proteomic analyses. Gels were stained with Coomassie brilliant blue for visualization and the entire protein-containing gel piece was processed for mass spectrometry as previously described41 (link).
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