Identical amounts of cell lysate (5–20ug) was mixed with 2× loading Laemmli buffer (Biorad) supplemented with DTT (Sigma) and incubated for 10 min at 95°C. Proteins were separated in 4–20% Tris-glycine polyacrylamide gels (Mini-PROTEAN; Bio-Rad) and electrophoretically transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 3% BSA in PBS and incubated with primary antibodies against APOBEC3A (PA5-78800 from Thermo Fisher) and/or APOBEC3B (E9A2G, Cell Signaling), as well as GAPDH (D4C6R, Cell Signaling). Secondary antibodies were goat anti-mouse IgG (H + L) DyLight 550 or goat anti-rabbit IgG (H + L) DyLight 650 (Thermo Fisher). After sequential washes in TBST buffer, fluorescent bands were visualized using a ChemiDoc Bio-Rad imager.
Goat anti mouse igg h l dylight 550
Manufactured by Thermo Fisher Scientific
Goat anti-mouse IgG (H + L) DyLight 550 is a secondary antibody conjugated with the DyLight 550 fluorescent dye. It is designed to detect and visualize mouse immunoglobulins (IgG) in various immunoassays and research applications.
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Lab products found in correlation
Dithiothreitol (dtt), by Merck Group (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Mini protean, by Bio-Rad (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Gapdh d4c6r, by Cell Signaling Technology (1 mentions)
Hoechst 33342 solution, by Thermo Fisher Scientific (1 mentions)
Anti phosphorylated neurofilament h nf h, by BioLegend (1 mentions)
2 protocols using goat anti mouse igg h l dylight 550
1
Immunoblotting of APOBEC3A and APOBEC3B
2
Immunocytochemical Characterization of Microgel-Cultured Neurons
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After 7 days in culture, neurons grown in microgels were collected in microcentrifuge tubes and washed with PBS. Cells were fixed with 1% glutaraldehyde for 1 min and washed 3x with PBS. Cells were blocked with 2% BSA/PBS at room temperature for 1 h and washed 3x with PBS. Cells were permeabilized with 0.1% Triton-X / 0.125% BSA/PBS and washed 3x with 0.125% BSA/PBS. The primary antibody, anti-Phosphorylated Neurofilament H (NF-H) (BioLegend), was added at 5 μg/mL, incubated at room temperature overnight, and then washed 3x in 0.125% BSA/PBS. The secondary antibody, Goat anti-Mouse IgG (H+L), DyLight 550 (#84540 ThermoFisher), and Hoechst 33342 Solution (ThermoFisher) were added at 5 μg/mL and 20 μg/mL respectively, for 1 h at 37 °C and then washed 3 times with 0.125% BSA/PBS. Cells and microgels were loaded onto glass slides and imaged on an epi-fluorescent microscope (Nikon Ti2). Cells were imaged in Phase Contrast/DAPI/RFP.
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