For amyloid plaques and ApoE western blots, equal volumes of FA extracted sample were subjected to 10–20% Tris-tricine gels (1mm, Novex) and 10% Tris-glycine gels, respectively, under denaturing conditions. After separation, proteins were transferred onto nitrocellulose membranes (Protran BA85; GE Healthcare), boiled in PBS for five min and blocked in I-Block solution (0.2% Tropix I-Block (Applied Biosystems), 0.1% Tween-20 in PBS) for one hour at room temperature with agitation. To detect different brain cell types from microglia enriched and depleted lysates, 1μg of total protein was loaded on 12% Tris-glycine gels and run under denaturing conditions. Proteins were subsequently transferred onto polyvinylidene difluoride membranes (PVDF Immobilon-P; Merck Millipore) and blocked in I-Block solution for one hour at room temperature. Primary antibodies (HJ6.3, Murine ApoE; 6E10, Aβ1–16; IBA1; GFAP; Tuj1; CNPase; HJ15.7, Human ApoE) were diluted in I-Block solution or TBS-Tween and incubated overnight at 4°C with agitation. Blots were washed with TBS-Tween and incubated in corresponding HRP-conjugated secondary antibodies for one hour at room temperature and visualized using enhanced chemiluminescence technique (Pierce).
Tris tricine gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
Tris-Tricine gels are a type of polyacrylamide gel electrophoresis (PAGE) system used for the separation and analysis of proteins, particularly low-molecular-weight proteins. These gels utilize a Tris-Tricine buffer system, which provides improved resolution and separation of proteins compared to traditional Tris-glycine gels. Tris-Tricine gels are commonly used in applications such as protein profiling, molecular weight determination, and analysis of protein expression patterns.
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Lab products found in correlation
Horseradish peroxidase conjugated goat anti mouse secondary antibody, by Cayman Chemical (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Protran ba85, by GE Healthcare (2 mentions)
Tropix 1 block, by Thermo Fisher Scientific (2 mentions)
Immobilon p pvdf, by Merck Group (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Protein assay, by Bio-Rad (1 mentions)
Amersham ecl plus, by GE Healthcare (1 mentions)
Protran, by Cytiva (1 mentions)
L 685 458, by Merck Group (1 mentions)
Gelcode, by Thermo Fisher Scientific (1 mentions)
Anti aβ antibody 6e10, by Fortrea (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Thermo scientific supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Colloidal blue, by Thermo Fisher Scientific (1 mentions)
Bs3 crosslinker, by Thermo Fisher Scientific (1 mentions)
Tricine gel, by Thermo Fisher Scientific (1 mentions)
11 protocols using tris tricine gel
1
Western Blot Analysis of Amyloid Plaques and ApoE
2
Western Blot Analysis of Amyloid Plaques and ApoE
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For amyloid plaques and ApoE western blots, equal volumes of FA extracted sample were subjected to 10–20% Tris-tricine gels (1mm, Novex) and 10% Tris-glycine gels, respectively, under denaturing conditions. After separation, proteins were transferred onto nitrocellulose membranes (Protran BA85; GE Healthcare), boiled in PBS for five min and blocked in I-Block solution (0.2% Tropix I-Block (Applied Biosystems), 0.1% Tween-20 in PBS) for one hour at room temperature with agitation. To detect different brain cell types from microglia enriched and depleted lysates, 1μg of total protein was loaded on 12% Tris-glycine gels and run under denaturing conditions. Proteins were subsequently transferred onto polyvinylidene difluoride membranes (PVDF Immobilon-P; Merck Millipore) and blocked in I-Block solution for one hour at room temperature. Primary antibodies (HJ6.3, Murine ApoE; 6E10, Aβ1–16; IBA1; GFAP; Tuj1; CNPase; HJ15.7, Human ApoE) were diluted in I-Block solution or TBS-Tween and incubated overnight at 4°C with agitation. Blots were washed with TBS-Tween and incubated in corresponding HRP-conjugated secondary antibodies for one hour at room temperature and visualized using enhanced chemiluminescence technique (Pierce).
3
Protein Extraction and Western Blot Analysis
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Cells were washed and trypsinized, and the resulting pellet was lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate; 50 µl for ~200,000 cells) and treated with 0.2 µl benzonase (250 U µl−1) per 50 µl of lysate at 37 °C for 10–20 min. Protein concentration was determined using the Bio-Rad protein assay. Total protein lysate (5–20 µg) was used for the western blot. The electrophoresis was run at 30 mA per minigel or at 120–180 V per camera.
For Pol II blots, home-made 6% acrylamide gels (37.5:1 acrylamide:bis-acrylamide ratio) were prepared and run for 1 h at 30 mA per minigel and transferred to 0.4-µm nitrocellulose membranes in standard Laemmli TB for 2 h at 200 mA with an ice block.
For histone blots, premade 12% Bolt Tris–tricine gels (Novex) were run in proprietary Novex MES running buffer according to the manufacturer’s manual and transferred to 0.2-µm nitrocellulose membranes in Novex transfer buffer for 1 h at 200 mA with an ice block.
After transfer, all membranes were blocked with 2% milk in TBS-T (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20), incubated overnight with primary antibodies (Supplementary Table2 ), washed three times with TBS-T, incubated for 30–60 min with secondary antibodies and developed using the Odyssey infrared scanner.
For Pol II blots, home-made 6% acrylamide gels (37.5:1 acrylamide:bis-acrylamide ratio) were prepared and run for 1 h at 30 mA per minigel and transferred to 0.4-µm nitrocellulose membranes in standard Laemmli TB for 2 h at 200 mA with an ice block.
For histone blots, premade 12% Bolt Tris–tricine gels (Novex) were run in proprietary Novex MES running buffer according to the manufacturer’s manual and transferred to 0.2-µm nitrocellulose membranes in Novex transfer buffer for 1 h at 200 mA with an ice block.
After transfer, all membranes were blocked with 2% milk in TBS-T (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20), incubated overnight with primary antibodies (Supplementary Table
4
Protein Expression Analysis in Mycobacteria
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Strains were inoculated from frozen stocks into 7H9 medium and grown to saturation. They were then diluted 1:500 in chelated Sauton’s medium, grown to saturation, and diluted 1:100 in chelated Sauton’s medium. Proteins from cell pellets and supernatants of cultures grown for 12 h in this fashion were run on 10 to 20% Tris-Tricine gels (Invitrogen) and revealed using an anti-FLAG antibody. An antibody to RNA polymerase (RNAP; Neoclone W0023), an intracellular protein, served as a loading and lysis control.
5
Western Blot Analysis of Amyloid-Beta in CSF and Brain
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CSF and Brain homogenates used for injection and immunoprecipitation were subjected to SDS‐PAGE using 10%–20% Tris‐tricine gels (Invitrogen). Proteins were transferred onto a nitrocellulose membrane (0.1‐µm pore size; Protran; Whatman) and probed with antibodies specific to human Aβ (6E10, Covance, 1:2000 dilution), and (3552; 1:2000 dilution) or with an antibody specific to oligomers (A11, Millipore, 1:1000 dilution) and visualized using Amersham ECL plus (GE Healthcare).
6
Analyzing γ-Secretase Activity in PS1 Mutants
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Membrane fractions of PS1/2−/− MEF cells stably transduced with PS1 WT or PS1 L435F were prepared as described (Sastre et al, 2001 ) and solubilized with 1% CHAPSO. Following a clarifying spin (100,000 × g, 30 min, 4°C), γ‐secretase activity was assessed using recombinant 1.4 μM C100‐His6 substrate (Edbauer et al, 2003 ) in assay buffer (150 mM sodium citrate pH 6.4, 0.5 mg/ml phosphatidylcholine, 10 mM DTT, 0.1 mg/ml BSA, 0.25% CHAPSO, 1 × PI) in the presence or absence of 0.5 μM L‐685,458 (Merck Millipore). Samples were separated by SDS–PAGE on 10‐20% Tris‐Tricine gels (Invitrogen), and AICD generation was analyzed by immunoblotting using antibodies Penta‐His or anti‐AICD Val50.
7
Photocrosslinking of RNA Polymerase
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For photo-crosslinking using σD581BpA and wt core in Figure 3 , 38 pmol of σ (in 1 μl TGED/NaCl/Triton) were incubated with 1 μl AsiA buffer [10 mM Tris-Cl (pH 8), 0.1 mM EDTA, 50% glycerol, 500 mM NaCl, 0.1 mM DTT] with or without 65 pmol AsiA and 1 μl of 5× Kglu transcription buffer lacking BSA [40 mM Tris-acetate (pH 7.9), 150 mM potassium glutamate, 4 mM magnesium acetate, 0.1 mM EDTA, and 0.1 mM DTT] for 10 min at 37°C in 1.5 ml eppendorf tubes. RNAP core (4 pmol in 2 μl RNAP storage buffer) or buffer alone was then added and the solution incubated for 10 min at 37°C before collection on ice. Tubes were laid flat on the UV lamp for a total of 30 min at room temperature, turning the tubes to the opposite side after 15 min. Samples were electrophoresed on 10–20% Tris-tricine gels (Invitrogen) and stained with Colloidial Coomassie Blue (Invitrogen). Photocrosslinking reactions in Figure 6B were assembled similarly except reactions contained 120 pmol σD581BpA, 400 pmol AsiA, and 15 pmol of wt or mutant core in a total volume of 30 μl. A 20 μl aliquot was used for photocrosslinking, and a 5 μl aliquot was applied to a 4–12% Tris-glycine gel (Invitrogen/Thermo Fisher) run in 1× Native Tris-glycine buffer (Invitrogen/Thermo Fisher) and stained in Gel Code (Thermo Fisher) as described (46 (link)).
8
Amyloid-beta detection by western blot
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The Aβ samples from in vivo or in vitro experiments were analysed by gel electrophoresis followed by western blotting using an anti-Aβ antibody (6E10)24 (link)25 27 (link)28 (link)29 (link). Samples (10 μl) were separated on a 10–20% Tris-tricine gel (Invitrogen, Grand Island, NY, USA). Following separation, the proteins were transferred onto nitrocellulose membranes and blocked with bovine serum albumin (BSA, 3% w/v, Sigma-Aldrich, St Louis, MO, USA) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 2 h at room temperature or overnight at 4 °C. The membranes were incubated with an anti-Aβ antibody (6E10, 1:2,000, Covance, Princeton, NJ, USA) in a solution of 2% BSA (w/v in TBS-T) for 4 h at room temperature or overnight at 4 °C. After washing with TBS-T (3 × , 10 min), a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:5,000 in 2% BSA w/v in TBS-T; Cayman Chemical Company, Ann Arbor, MI, USA) was added for 1 h at room temperature. The Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA), Biosesang ECL Plus kit (Biosesang, Gyeonggi-do, Republic of Korea), or a homemade ECL kit53 (link) was used to visualize the results on a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).
9
Amyloid-Beta Protein Analysis by Western Blot
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The samples from the inhibition and disaggregation experiments were analyzed by gel electrophoresis with Western blot using an anti-Aβ antibody (6E10)20 (link)28 (link). Each sample (10 μL) was separated on a 10–20% Tris-tricine gel (Invitrogen, Grand Island, NY, USA). Following separation, the gel was transferred onto nitrocellulose membrane which was blocked with bovine serum albumin (BSA, 3% w/v, Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 3 h at room temperature. The membrane was treated with antibody (6E10, Covance, Princeton, NJ, USA; 1:2000) in a solution of BSA (2% w/v) in TBS-T overnight at 4 °C. Following washing, the membrane was treated with horseradish peroxidase-conjugated goat antimouse secondary antibody (1:5000; Cayman Chemical, Ann Arbor, MI, USA) in 2% BSA in TBS-T solution for 1 h at room temperature. Protein bands were visualized using ThermoScientific Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
10
Western Blot Analysis of Amyloid-β Aggregation
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