The pSNAP-tag(m) plasmid, which was used as backbone, is available at Addgene (#101135). The transfection reagent TransIT-LT-1 was purchased from Mirus (Lot81023060) and the ncAA TCO*K (axial trans-cyclooct-2-ene–L–lysine; CAS 1801936-26-4) was purchased from SiChem. We used four fluorescent dyes for labeling. The SNAP-Cell® 647-SiR (S9102 S) and the SNAP-Cell® TMR-Star (S9105 S) were purchased from New England BioLabs and the 6-methyl-tetrazine-BDP-FL is produced by Jena Bioscience (RK011-001). The SiR-DNA kit (SC007) was purchased by Spirochrome. For detecting Aβ, we used the High sensitive IBL-ELISA: Amyloid-beta (1–42) (LOT. 1D-818) and Amplex® UltraRed from Molecular Probes, Thermo Fisher Scientific. The 4–20% polyacrylamide Bis-Tris gels were purchased by BioRad. The cOmplete™, EDTA-free Protease Inhibitor Cocktail was purchased from Sigma (000000005056489001). The primary antibodies we used are the Anti-SNAP-tag® antibody (Polyclonal), (1 : 200) from New England BioLabs (P9310 S) and the Anti-β-Amyloid, 1–16, Clone: 6E10 (1 : 1000) from BioLegend (803001). The secondary antibodies are the IRDye 680RD Donkey anti-Rabbit (926-68073) and IRDye 800 CW Donkey anti-mouse (92632212) from LI-COR Biosciences.
Polyacrylamide bis tris gels
Manufactured by Bio-Rad
The 4–20% polyacrylamide Bis-Tris gels are laboratory equipment used for protein separation and analysis. They are designed to provide a linear separation range for proteins within the specified molecular weight range.
Automatically generated - may contain errors
Lab products found in correlation
Sir dna kit, by Spirochrome (1 mentions)
Amplex ultrared, by Thermo Fisher Scientific (1 mentions)
Anti snap tag antibody polyclonal, by New England Biolabs (1 mentions)
Irdye 680rd donkey anti rabbit, by LI COR (1 mentions)
Irdye 800cw donkey anti mouse, by LI COR (1 mentions)
Snap cell tmr star, by New England Biolabs (1 mentions)
Complete edta free protease inhibitor cocktail, by Merck Group (1 mentions)
Transit lt1, by Mirus Bio (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
2 protocols using polyacrylamide bis tris gels
1
Labeling Amyloid-beta with Fluorescent Probes
2
SiR-tetrazine Labeling and Western Blot Analysis
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Cells were lysed in N-Ripa Buffer (10 mM Tris-HCl (pH 7.6), 1 mM EDTA, 0.1% sodium dodecyl sulfate, 0.1% sodium deoxycholate, 1% Triton X-100, 5% Glycerol, 140 mM NaCl, 1x protease inhibitor) and sonicated for 20 cycles (30 sec on/30 sec off). Protein concentration was determined by BCA assay (Thermofisher, 23227). For lysate labeling 1 µM SiR-tetrazine (Spirochrome, SC008) was added to the lysate for 5 min. SDS PAGE sample buffer was added and samples were boiled briefly at 95℃. 25 μg protein were loaded in each well. Samples were loaded on 4-20% polyacrylamide Bis-Tris gels (BioRad, #4561096) and exposed for in-gel fluorescence at 460nm and 630 nm. The protein was transferred to nitrocellulose membranes and probed with FLAG-Antibody and GAPDH antibodies and imaged with the GE Healthcare ImageQuant LAS 500.
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