Cerebrum samples from eight-week-old mice were homogenized and divided into four subcellular fractions enriched with cytoplasmic (C), membrane (M), nuclear (N), or insoluble (I) proteins using EzSubcell Extract (WSE-7421, ATTO, Tokyo, Japan). The fractions (C, N, and I), 3 µg per lane, were subjected to western blotting as described previously41 (link). The blots were subsequently incubated with the following antibodies: TDP-43 (1:2000, 10782-2-AP, Proteintech, Chicago, IL, USA), α-tubulin (1:5000, T5168, Sigma-Aldrich), or p84 (1:1000, ab131268, Abcam, Cambridge, UK). For subcellular fractionation of cultured cells, the Pierce NE-PER reagents (Thermo Fisher Scientific) were applied according to the method used by Nonaka et al.24 (link), and antibodies for p84 (1:1000, GTX70220, GeneTex, Irvine, CA, USA) and GFP (1:1000, sc-9996, Santa Cruz Biotechnology, Dallas, TX, USA) were used for detection.
Gtx70220
Manufactured by GeneTex
Sourced in United States
The GTX70220 is a microplate reader designed for high-throughput analyses. It can measure absorbance, fluorescence, and luminescence in standard 96-well and 384-well microplates. The device is capable of reading multiple wavelengths and is suitable for a variety of applications, including enzyme-linked immunosorbent assays (ELISA), cell-based assays, and nucleic acid quantification.
Automatically generated - may contain errors
Lab products found in correlation
Ap160p, by Merck Group (2 mentions)
Ab131268, by Abcam (1 mentions)
Sc 9996, by Santa Cruz Biotechnology (1 mentions)
T5168, by Merck Group (1 mentions)
Gtx127277, by GeneTex (1 mentions)
Gtx128092, by GeneTex (1 mentions)
Ab93806, by Abcam (1 mentions)
Phospho creb, by Cell Signaling Technology (1 mentions)
Pparα, by Santa Cruz Biotechnology (1 mentions)
Rev erbα, by Cell Signaling Technology (1 mentions)
Per2 per21 a, by Alpha Diagnostic (1 mentions)
Foxo1, by Cell Signaling Technology (1 mentions)
Sirt1, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg antibody, by Jackson ImmunoResearch (1 mentions)
Mini protean tgx stain free, by Bio-Rad (1 mentions)
Trichostatin a, by Merck Group (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
5 protocols using gtx70220
1
Subcellular Fractionation and Western Blotting of Mouse Brain
2
Immunoblot Analysis of DENV Infection
3
Antibodies for Circadian Clock Proteins
4
Western Blot Analysis of Lamin A/C in HGPS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western Blot analysis was performed on HGPS patient B-lymphoblast cells obtained from the Coriell Institute (Affected HGPS patient; AG03506, unaffected sibling; AG03507). Fresh B-lymphoblast cell culture pellets were supplemented with 8 M UREA, 5% RIPA buffer including protease and phosphatase inhibitors (Roche, according to manufacturers guidelines) and homogenized by pipetting and vortexing. The protein lysates were briefly sonicated (2 min. at 4 °C on high amplitude) and the nuclear fraction was isolated using the Minute Nuclear Envelope Extraction Kit (NE-013). The isolated nuclear fractions were loaded on a SDS-PAGE gel (4% Mini-PROTEAN TGX Stain-Free, 456-8036 Bio-Rad) and transferred on a nitrocellulose membrane (1704158, Bio-Rad). The membrane was incubated with primary antibodies against lamin A/C (1:200, sc-376248 E-1, Santa Cruz Biotechnology) and the nuclear protein p84 (1:500, GTX70220 GeneTex) in 5% TBS-T milk over night at 4 °C. Secondary antibody staining was performed on the following day with a HRP-conjugated goat anti-mouse IgG antibody (1:10000, Jackson-ImmunoResearch).
5
Western Blot Analysis of BMAL1 and P84
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen tissue was added to RIPA buffer (50 mM Tris-HCl [pH 8.0], 1% NP-40, 0.1% SDS, 150 mM NaCl, 5 mM MgCl2, 0.5% sodium deoxycholate, and 1 mM PMSF) supplemented with 330 nM trichostatin A (Sigma), Protease Inhibitor Cocktail (Roche), 20 mM NaF, 1 mM DTT, and 10 mM nicotinamide and then lysed with 10 sec of sonication at 60% power using a VWR 200 homogenizer (Avantor). The resulting lysates were centrifuged for 15 min at 13.2 10 6 g at 4°C, and the supernatant was retained. Protein concentrations were quantified by Bradford assay (Bio-Rad). Next, 10-20 μg of protein was separated in an 8% polyacrylamide gel and transferred to nitrocellulose membranes, which were incubated with anti-BMAL1 (Abcam, ab93806) or anti-P84 (Genetex, GTX70220) overnight at 4°C. The membranes were subsequently incubated with peroxidase-conjugated secondary antibodies (anti-mouse IgG, HRP conjugate, EMD Millipore, AP160P; anti-rabbit IgG, HRP-linked, EMD Millipore, 12-348) at room temperature for 1 hour, and then visualized using chemiluminescent HRP substrate (WBKLS0500, EMD Millipore).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!