MDA-MB-231 and SH-SY5Y cells were lysed with protein lysis buffer MPER (Thermo Fisher Scientific Inc., Waltham, MA) supplemented with a protease inhibitor cocktail (P2714, Sigma) and centrifuged at 10,000 g for 15 min at 4 °C. The supernatant was incubated with anti-NCS-1 antibody (FL190, Santa Cruz Biotechnology, Santa Cruz, CA, 1: 5000) for 1 h at 4 °C and then precipitated employing protein-A magnetic beads (PureProteome, EMD Millipore, Billerica, MA) by incubating for 1 h at 4 °C, following the manufacturer's instructions. Beads were washed and then eluted, and protein levels were quantified using western blotting, employing anti-NCS-1 (FL190, Santa Cruz Biotechnology, Santa Cruz, CA, 1: 10,000) or anti-TRPA1 (Alomone, Jerusalem, Israel, 1:500) antibodies. Co-IP experiments were also conducted in reverse mode, employing anti-TRPA1 (Alomone, Jerusalem, Israel, 1:500) to induce immunoprecipitation and then using anti-NCS-1 and anti-TRPA1 in western blot analysis to assess the interactions between NCS-1 and TRPA1.
Anti trpa1
Manufactured by Alomone
Sourced in Israel
Anti-TRPA1 is a laboratory equipment product that is designed to detect and measure the TRPA1 protein. TRPA1 is a member of the transient receptor potential (TRP) ion channel family and plays a role in various physiological processes. This product can be used in research applications to study the expression and function of TRPA1 in different cell types or experimental models.
Automatically generated - may contain errors
Lab products found in correlation
M per, by Thermo Fisher Scientific (2 mentions)
Pureproteome, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Pgc 1α, by Abcam (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Anti trpa1, by Novus Biologicals (1 mentions)
Anti β actin, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Agilent Technologies (1 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Agilent Technologies (1 mentions)
Mapk family antibody sampler kit, by Cell Signaling Technology (1 mentions)
Phospho mapk family antibody sampler kit, by Cell Signaling Technology (1 mentions)
Anti α actinin, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Alexa fluor 594 affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 affinipure goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
β actin, by Bio-Rad (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
P2714, by Merck Group (1 mentions)
4 protocols using anti trpa1
1
Co-Immunoprecipitation of NCS-1 and TRPA1
2
Western Blot Protein Detection Protocol
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Western blot was done as previously described [19 (link)–21 (link)]. Primary antibodies used in the study were as follows: anti-TRPA1 (1:1000, Alomone), anti-TRPA1 (1:1000, Novus Biologicals, CO, USA), anti-TRPA1 (1:1000, LSBio, WA, USA), MAPK Family Antibody Sampler Kit (1:1000, Cell Signaling Technology), Phospho-MAPK Family Antibody Sampler Kit (1:1000, Cell Signaling Technology), anti-MKP-1 (1:1000, Thermo Fisher Scientific), PGC-1α (1:1000, Abcam), anti-β-actin (1:1000, Abcam), anti-β-tubulin (1:1000, Cell Signaling Technology). Secondary antibodies used were: HRP-conjugated goat anti-rabbit secondary antibody (1:3000, Dako), HRP-conjugated goat anti-mouse secondary antibody (1:3000, Dako).
3
Immunocytochemical Analysis of TRPA1
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Primary antibodies used in the study were: anti-TRPA1 (1:200, Alomone, Jerusalem, Israel), anti-α-actinin (1:200, Abcam, Cambridge, UK). Secondary antibodies used were: Alexa Fluor 405 goat anti-mouse IgG (1:200, Alexa), Alexa Fluor 488 goat anti-mouse IgG (1:200, Thermo Fisher Scientific), Alexa Fluor 488 AffiniPure goat anti-rabbit IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA), Alexa Fluor 594 AffiniPure goat anti-mouse IgG (1:200, Jackson ImmunoResearch). Cell imaging was performed with Leica SP8 confocal microscope (Leica, Wetzlar, Germany) and analyzed by Fiji (NIH).
4
Immunoblotting of TRPA1, NCS-1, and β-actin in MDA-MB-231 cells
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Cultured MDA-MB-231 cells were lysed with protein lysis buffer MPER (Thermo Fisher Scientific Inc., Waltham, MA) supplemented with a protease inhibitor cocktail (P2714, Sigma Aldrich, 1:100) and centrifuged at 10,000 g for 15 min at 4 °C. Cell lysates were separated by SDS–PAGE, followed by electrophoretic transfer onto PVDF membranes. The membranes were cut into three sections, where each section included the molecular weight of the target protein, and each segment was incubated with the corresponding primary antibody. The primary antibodies used were anti-TRPA1 (Alomone, Jerusalem, Israel, 1:500) (Alomone Labs Cat# ACC-037, RRID:AB_2040232), anti-NCS-1 (FL190, Santa Cruz Biotechnology, Santa Cruz, CA, 1:10,000) (Santa Cruz Biotechnology Cat# sc-13037, RRID:AB_649907), and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, 1:2000) (Santa Cruz Biotechnology Cat# sc-130301, RRID:AB_2223360). Membranes were incubated with primary antibodies overnight at 4 °C. The bands were visualized by an enhanced chemiluminescence system after incubation with rabbit secondary antibody (1:20,000) (Bio–Rad Cat# 166–22,408 EdU) for TRPA1 and NCS-1 and mouse secondary antibody (1:20,000) (Bio–Rad Cat# 170–6516) for β-actin for 2 h at room teMPERature.
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