For protein sample preparation, cells were washed in PBS, harvested in 8 M urea, and subjected to sonication. The protein concentration in the lysates was determined by the Bradford method. The primary antibodies used for detection of the respective proteins by SDS-PAGE and Western blotting were anti-annexin VI antibody (N-19; Santa Cruz catalog no. sc-1931), anti-GAPDH antibody (FL-335; Santa Cruz catalog no. sc-25778), anti-IFITM3 antibody (Proteintech; 11714-1-AP), anti-IFITM2/3 antibody (Proteintech; 66081-1-Ig), anti-influenza M1 antibody (clone GA2B; AbD Serotec [MCA401]), and anti-α-tubulin antibody (clone B-5-1-2; Sigma catalog no. T5168). IRDye secondary antibodies (Li-COR) labeled with near-infrared (NIR) fluorescent dyes were used for direct, nonenzymatic detection of primary antibodies coupled with IRDye 680CW and IRDye 800CW for donkey anti-mouse IgG (H+L), donkey anti-rabbit IgG (H+L), and donkey anti-goat IgG (H+L). An Odyssey infrared imaging system (Li-COR) was used for NIR fluorescence detection. Western blots were quantified using Odyssey infrared imaging system software version 3.0.25.
Anti ifitm3 antibody
Manufactured by Proteintech
The Anti-IFITM3 antibody is a research tool designed to detect the IFITM3 protein, which is involved in cellular processes such as viral entry and membrane trafficking. This antibody can be used in various techniques, including Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of IFITM3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (2 mentions)
Anti gapdh antibody fl 335, by Santa Cruz Biotechnology (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Anti α tubulin antibody clone b 5 1 2, by Merck Group (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti ha antibody, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti sars cov 2 n antibody, by Sino Biological (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Proteintech (1 mentions)
Cm100, by Philips (1 mentions)
Goat anti mouse igg gold antibody, by Merck Group (1 mentions)
3 protocols using anti ifitm3 antibody
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of SARS-CoV-2 and IFITM3
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Cells were lysed in RIPA buffer (25 mM Tris /HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA). After clarification by centrifugation, equal amounts of cell lysates quantified by BCA™ Protein Assay Kit (Thermo Fisher Scientific, MA, USA) were separated in SDS-PAGE and transferred to nitrocellulose membranes. Then, membranes were probed with the indicated antibodies, anti-HA antibody from Sigma-Aldrich (St. Louis, MO, USA) (1:4000, Cat. No. H6908), anti-Flag antibody from Sigma-Aldrich (1:4000, Cat. No. F3165), anti-actin antibody from Sigma-Aldrich (1:5000, Cat. No. A1978), anti-SARS-CoV-2 N antibody from Sino Biological (1:1000, Cat. No. 40143-R019), and anti-IFITM3 antibody from Proteintech (1:1000, Cat. No. 11714-1-AP), followed by IRDye™ secondary antibodies (Odyssey, Lincoln, NE, USA). The signals were collected with the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA).
3
Immunogold Labeling of IFITM3 in Viral Particles
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Viral particles were purified as described above. Carbon-coated nickel grids were glow discharged before purified viral particle solution was applied. Samples were fixed with 4% paraformaldehyde in 0.1 M Hepes for 7 min. After one short wash in PBS, samples were permeabilized with 0.5% Triton X-100 in PBS for 1 min and washed again three times in PBS. Free aldehydes were quenched with 0.15% glycine in PBS. To prevent unspecific antibody binding, samples were blocked in 1% BSA (Merck) in PBS. Primary antibody staining was performed using rabbit polyclonal anti-IFITM3 antibody (Proteintech) diluted in 1% BSA in PBS. Samples were washed three times in 1% BSA in PBS, before secondary antibody staining was performed using a goat anti-mouse IgG Gold antibody (Sigma-Aldrich) at an OD of 0.15. After secondary antibody staining, samples were washed in PBS and fixed in 4% paraformaldehyde in 0.1 M Hepes, before being negatively stained with 1% uranyl acetate and imaged in a Philips CM100 transmission electron microscope operated at 100 kV.
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