At 48 h after transfection, cells were lysed in TNT-G buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% Triton-X100 with protease inhibitor cocktail (Roche). After 30 min incubation on ice, the cell suspension was centrifuged (20,000 g for 20 min) and the supernatant was collected. The cell lysates were incubated with anti-FLAG M2 agarose affinity gel (Sigma) at 4 °C overnight and were eluted with 4% SDS sample buffer (27 (link)). For the analysis of ER stress markers, the samples of subcellular fraction were used. Samples were resolved by SDS-PAGE and transferred to a PVDF membrane (Polyscreen, PerkinElmer, Boston, MA). A western blot image was obtained using a chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL).
Polyscreen
Manufactured by PerkinElmer
Sourced in Australia
Polyscreen is a high-performance membrane filtration device designed for laboratory applications. It facilitates the separation and concentration of macromolecules, cells, and other particulates from liquid samples. The product's core function is to enable efficient and reliable filtration processes in various research and analytical settings.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence detection kit, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by PerkinElmer (2 mentions)
Novex ecl chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (2 mentions)
Xcell surelock mini cell system, by Thermo Fisher Scientific (2 mentions)
Las 4000, by Cytiva (2 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti flag m2 affinity agarose gel, by Merck Group (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Rat anti tdp 43 2h4, by BioLegend (1 mentions)
Anti tdp 43 polyclonal, by Proteintech (1 mentions)
Anti p65 polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Anti p65, by Thermo Fisher Scientific (1 mentions)
Dyanl, by Thermo Fisher Scientific (1 mentions)
Rabbit anti alix, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Goat anti rabbit igg, by Cytiva (1 mentions)
8 protocols using polyscreen
1
Affinity Purification and Western Blot Analysis
2
Western Blot Analysis of Itm2a in CD4+ Cells
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In each sample, 1×106 CD4+ cells were lysed in freshly prepared radioimmumoprecipitation assay buffer. Cell lysate was separated from debris by centrifugation at 12,000 rpm for 10 min. Lysate was loaded onto 12% polyacrylamide gels and transferred onto PVDF membrane (Polyscreen; Perkin Elmer). The membrane was subsequently blocked in 5% milk and probed with Itm2a antibody. The rabbit anti-human Itm2a antibody was generated by using Itm2a extracellular domain as an immunogen. Proteins were visualized using an ECL kit (PerkinElmer).
3
Western Blot Protein Analysis
4
Western Blot Analysis of Ets-1 and CCR7
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In each sample, cells were lysed with freshly prepared whole-cell lysis buffer. Cell lysate was separated from debris by centrifugation at 13,000 rpm for 10 min. The lysate was loaded onto 10% polyacrylamide gels and transferred onto PVDF membranes (PolyScreen, PerkinElmer). These membranes were subsequently blocked in 5% milk and probed with an anti-Ets-1 or anti-CCR7 antibody. Proteins were visualized using an enhanced chemiluminescence (ECL) kit (PerkinElmer).
5
TDP-43 Overexpression Immunoblot Assay
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N2a cells have been transfected with pFN33_TDP-43 and pFN35_TDP-43 construct as described above and incubated for 24 h at 37 °C. Cells have been lysed with 500 µl Laemmli lysis buffer per 2 wells of a 12-well plate (Tris/HCl, pH 6.8, 62.5 mM, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.1 M DTT). Proteins were separated using a 12% SDS-PAGE and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (PolyScreen; PerkinElmer Life Sciences, Boston, MA). Membranes were washed once in TBS-T buffer (150 mM NaCl, 20 mM Tris/HCl pH 7.4, 0.05% Tween 20), blocked in TBS-T containing 1% BSA (w/v), and probed with rat anti-TDP-43 2H4 (1:2000, BioLegend, San Diego, CA, USA). Detection of bound primary antibody was performed with rabbit anti-rat antibody (1:10000, DAKO GmbH, Jena, Germany). Blots were developed with ECL reagents and imaged in ECL-Imager (DNR MF-ChemiBIS Image Analysis System 1.6, DNR Bio-Imaging Systems, Biostep, Germany).
6
Quantification of TDP-43, p65, and Tau Proteins
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TDP-43, p65 and tau were quantified using Western immunoblot. Proteins (15 μg/sample) were heated at 95°C for 5 minutes in SDS sample buffer. For immunoprecipitation study, anti-TDP-43 polyclonal (ProteinTech, Chicago) or anti-p65 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz) was bound to protein G-coated magnetic beads (Dyanl, Invitrogen, Camarillo) and was incubated with 50 μg of lysate overnight at 4°C. After washing, immunoprecipitates were eluted with SDS sample buffer. Samples were resolved by 10% SDS-PAGE and transferred to a PVDF membrane (Polyscreen, PerkinElmer, Boston, MA). The membrane was incubated with anti-p65 (Invitrogen, Camarillo) or anti-TDP-43 2E2-D3 antibody (Abnova, Walnut), and western blot image was obtained using a chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL). Each protein was estimated by standardization with actin.
7
Protein Immunoblotting Analysis Protocol
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Analyzed proteins were quantified and used for immunoblotting analyses. The equal quantified proteins or equal volumes of immunoprecipitated proteins were analyzed by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes (PolyScreen, PerkinElmer). Transferred antigens were incubated with the following antibodies: mouse anti-galectin-3, rabbit anti-Alix, mouse anti-HIV-1 p24 (Millipore, Burlington, MA, USA), mouse anti-β-actin (Sigma, St. Louis, MO, USA), or rabbit anti-α-tubulin (Epitomics, Burlingame, CA, USA), and held for 1 h at 37 °C. After three washes with 1x phosphate-buffered saline with 0.1% Tween® detergent (PBST), membranes were incubated with horseradish peroxidase (HRP)-conjugated antibodies (goat anti-mouse IgG, goat anti-human IgG, or goat anti-rabbit IgG) (Amersham Biosciences, Charlemagne, UK) for 1 h at 37 °C. Hybridized protein bands were created with an Immobilon™ Western Enhanced Chemiluminescence (ECL) protein detection system (Millipore, Burlington, MA, USA).
8
Western Blot Quantification Protocol
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Ten micrograms of total cell protein from whole-cell lysates was heat-denatured (5 min at 95 °C in NuPAGE® LDS Sample Buffer and reducing agent), then electrophoresed at 130 V for 1 h using pre-cast gels in an XCell SureLock Mini-Cell system (Life Technologies). The protein was then transferred to polyvinylidene difluoride membranes (0.2 μm Polyscreen®, PerkinElmer). The transfer membranes were blocked for 1 h at RT using a 5% (w/v) skim milk solution (for clone 3A11) in 0.1% (v/v) TBS-Tween®-20 (blocking solution) or 5% BSA (w/v; for clone 2C6) and incubated with primary antibody overnight at 4 °C. The membranes were washed in 0.1% (v/v) TBS-Tween®-20 and then incubated with the appropriate HRP-conjugated secondary antibody diluted 1:10,000 in 5% milk block. Membranes were visualised using Novex® ECL chemiluminescent substrate reagent kit (Life Technologies) and ImageQuant™ LAS 4000 imager, software version 1.2.0.101 (GE Healthcare Pty Ltd., NSW, Australia). The intensity of the signal was quantified relative to a reference GAPDH loading control and Amido Black total protein staining (Sigma Aldrich) using AlphaViewSA™ software v3.0 (ProteinSimple Pty Ltd., CA, USA).
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