Cells extracts were prepared in PBS containing 1 × protease inhibitor (Roche Diagnostics, Indianapolis, IN, USA). Proteins were separated by SDS-PAGE and transferred to nitrocellulose (Cytiva, formerly GE Healthcare, Chicago, IL, USA) and protein detection was carried out with standard Western blotting techniques. After transfer, membranes were incubated for 10 h in blocking solution (5% nonfat dry milk in PBS containing 0.1% Tween-20, T-PBS) to prevent non-specific binding. Subsequently, membranes were incubated for 1 h at room temperature with specific primary antibodies diluted in blocking solution. Expression levels of both endogenous and flag-tagged human wild-type TDP-43 was monitored by using a commercially available polyclonal anti-TDP-43 antibody (Proteintech, Rosemont, IL, USA, 10782-2-AP). Endogenous tubulin was used as a loading control, using an in-house made mouse monoclonal antibody. Immunoblots were developed by using the ECL Star Enhanced Chemiluminescent Substrate (EuroClone, Pero, Milan, Italy).
Anti tdp 43 antibody
Manufactured by Proteintech
Sourced in United States
The Anti-TDP-43 antibody is a reagent used in scientific research. It is designed to specifically detect the TDP-43 protein, which is a DNA/RNA-binding protein involved in various cellular processes. The antibody can be utilized in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to study the expression and localization of TDP-43 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Protease inhibitor, by Roche (1 mentions)
Ecl star enhanced chemiluminescent substrate, by Euroclone (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Anti p62 antibody, by BD (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Alexa 488 conjugated anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Laemmli sds page sample buffer, by Bio-Rad (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Immobilon p polyvinylidene fluoride, by Merck Group (1 mentions)
Anti gfp antibody, by Cell Signaling Technology (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Blocking one reagent, by Nacalai Tesque (1 mentions)
6 protocols using anti tdp 43 antibody
1
Western Blot Analysis of TDP-43
2
Multimodal Analysis of Synucleinopathy
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Brain sections were stained with anti-pS129 α-syn as described above, with the exception that goat anti-rabbit secondary antibody conjugated with Alexa 647 (Thermo Fisher Scientific) was used (1:200 in blocking buffer) to avoid interference with LCO emission. PD sections were also stained with anti-α-syn antibody (Syn303, 1:1000, Biolegend), anti-phosphorylated tau antibody (AT100, 1:250, Thermo Fisher Scientific), anti-p62 antibody (1:100, BD Bioscience) and anti-TDP-43 antibody (1:500, Proteintech Group Inc.). After washing 3 × 5 min in PBS, they were incubated with 3 μM HS-68 for 30 min at room temperature. They were then washed with PBS and mounted with Dako mounting medium. Fluorescence images and emission spectra were collected using an inverted Zeiss LSM 780 confocal microscope.
3
Immunoprecipitation of FLAG-tagged TDP-43
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HeLa cells (70% of confluence) were transfected with 3 μg of pFLAG-TDP-43 wild-type using the Effectene reagent. After 24 h, cell culture medium was removed and cells were washed with cold PBS and harvested. Cells were lysed in 500 μl of IP buffer (20 mM Tris pH 7.5, 110 mM NaCl, 0.5% Triton-X, 1× Complete Protease Inhibitor Cocktail) by sonication (3 min, mid power), in ice-cooled sonicating bath (BioRuptor, Diagenode, Belgium). The cell lysate was pre-cleared by incubation with 30 μl Protein A/G PLUS agarose beads (Santa Cruz Biotechnology Inc., Dallas, Texas, USA) in IP buffer for 1.5 h at 4°C. The pellets were discarded and the supernatants were used for immunoprecipitation: the cell lysates were incubated with 2 μg of mouse monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) on a rotating device for an hour at 4°C. Then, 30 μl of Protein A/G PLUS agarose beads were added to each sample and incubated overnight at 4°C. The pellet was then washed three times in ice-cold IP buffer. The supernatants was discarded, and the pellet was re-suspended in 30 μl of 3× sample loading dye. The samples were fractionated by SDS-PAGE (10%) and analyzed by immunoblotting 1:2000 rabbit polyclonal anti-TDP-43 antibody (ProteinTech), with 1:500 rabbit polyclonal anti-DAZAP1 antibody and 1:500 rabbit polyclonal anti-hnRNP H antibody previously described (39 (link),40 (link)).
4
Retinal Tissue Analysis in Female Flies
5
Sequential Biochemical Fractionation of Cells
6
Western Blot and Immunocytochemistry for TDP-43
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The samples were mixed with equal volume of 2x Laemmli SDS-PAGE sample buffer (Biorad) and boiled at 95 °C for 5 min. The samples were next subjected to SDS-PAGE and blotted onto Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore). Immunoblotting was performed by a standard protocol, and immunoreactive signals were detected by ChemiDoc Touch (Biorad) using ECL-select detection reagent (GE Amersham). The following antibodies were used; anti-TDP-43 antibody (Proteintech: 10782-2-AP); anti-GFP antibody (Cell Signaling: #2956); anti-GAPDH antibody (Cell Signaling: #2118).
Immunostaining HEK293 cells expressing D2-YFP, D1-TDP-43-YFP, or D2-TDP-43-YFP were treated with 0.5 μM AP20187 for 24 h and then fixed by formalin-PBS. The cells were permeabilized by 0.5% Triton-X100 and blocking was performed with Blocking-one reagent (Nacalai). Immunostaining was performed with anti-cleaved poly(ADPribose) polymerase-1 (PARP) antibody (Cell Signaling: #5625).
Immunostaining HEK293 cells expressing D2-YFP, D1-TDP-43-YFP, or D2-TDP-43-YFP were treated with 0.5 μM AP20187 for 24 h and then fixed by formalin-PBS. The cells were permeabilized by 0.5% Triton-X100 and blocking was performed with Blocking-one reagent (Nacalai). Immunostaining was performed with anti-cleaved poly(ADPribose) polymerase-1 (PARP) antibody (Cell Signaling: #5625).
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