Mosquito ovaries were dissected in 1× PBS under a dissecting microscope and homogenized in lysis buffer (12 mM sodium deoxycholate, 0.2% SDS, 1% triton X-100, complete mini EDTA-free protease inhibitor; Roche Applied Science, Indianapolis, IN, USA). Protein extracts were resolved on SDS-PAGE using a 12% acrylamide separation gel and a 3% stacking gel. The resolved proteins were either stained with GelCode Blue reagent (Thermo Fisher Scientific) or electrophoretically blotted to a nitrocellulose membrane (LI-COR, Lincoln, NE, USA) for western blot analysis. The membranes were blocked with 4% nonfat dry milk and incubated with each primary antibody in 4% nonfat milk in PBS containing 0.1% Tween 20. The EOF1 rabbit polyclonal antibody was generated by GenScript Corporation (Piscataway, NJ, USA) based on an antigenic peptide (LAPNSPSKEDEPAH). The anti-α-tubulin monoclonal antibody from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA) was used as loading controls for ovaries. The dilutions of the primary antibodies were as follows: EOF1 (1:3,000) and α-tubulin (1:2,000). The secondary antibodies were either IRDye 800CW goat anti-rabbit secondary antibody (1:10,000; LI-COR) or IRDye 800CW goat anti-mouse secondary antibody (1:10,000; LI-COR). The protein bands were visualized with an Odyssey Infrared Imaging System (LI-COR).
Gelcode blue reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
GelCode Blue reagent is a protein stain used for the detection of proteins in polyacrylamide gels. It provides a simple and sensitive method for visualizing proteins separated by gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Polyacrylamide gel, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by LI COR (1 mentions)
Irdye 800cw goat anti rabbit secondary antibody, by LI COR (1 mentions)
Irdye 800cw goat anti mouse secondary antibody, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Complete mini edta free protease inhibitor, by Roche (1 mentions)
Mops running buffer, by Thermo Fisher Scientific (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000c uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Precast gel, by Thermo Fisher Scientific (1 mentions)
Endoglycosidase h, by Roche (1 mentions)
Protein g agarose beads, by Repligen (1 mentions)
7 protocols using gelcode blue reagent
1
Mosquito Ovary Protein Extraction and Analysis
2
SDS-PAGE and Western Blot Analysis of VHHs
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VHHs were analyzed by reducing SDS-PAGE using NuPage Novex 4%-12% Bis-Tris gels with MOPS running buffer (Invitrogen) and staining with Gelcode Blue reagent (Thermo Fisher Scientific). Western blotting was performed by separating 2.5 µg TeNT on a single gel and electroblotting to a nitrocellulose membrane. After blocking the membrane with PBS containing 5% milk and 0.05% Tween-20, immunoblotting was performed in PBS containing 0.1% milk and 0.05% Tween-20 using 0.5 µg/ml biotinylated VHHs and 0.1 µg/ml streptavidin-HRP conjugate. Blots were visualized for HRP by enhanced chemiluminescence (LI COR Biosciences, USA).
3
Purification and Characterization of Tyrosinase
4
Single-domain Antibody Production in Yeast
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VHHs fused to the natural llama heavy-chain antibody long hinge region, and a hexahistidine (his6) tag was produced in baker’s yeast using vector pRL188 and purified from culture supernatant using immobilized-metal affinity chromatography as described earlier [23 (link)]. VHHs produced in this manner were indicated by the suffix “F.” About 10% of the VHH amount produced in this manner was dimerized through the single cysteine present at the C-terminus of the VHH, immediately preceding the his6 tag. Both monomeric and dimeric VHHs are useful for functional immobilization of VHHs to polystyrene surfaces by passive adsorption [9 (link),24 (link)]. Yeast-produced VHHs were biotinylated at a weight ratio of protein to biotin of 5 using amine-reactive sulfo-N-hydroxysuccinimide-LC-biotin (ThermoFisher Scientific).
VHHs were subjected to reducing SDS-PAGE using precast gels (Novex, San Diego, CA, USA), and stained using GelCode Blue reagent (ThermoFisher Scientific). VHHs were digested with endoglycosidase H (Roche Applied Science) according to the manufacturer’s instructions.
VHHs were subjected to reducing SDS-PAGE using precast gels (Novex, San Diego, CA, USA), and stained using GelCode Blue reagent (ThermoFisher Scientific). VHHs were digested with endoglycosidase H (Roche Applied Science) according to the manufacturer’s instructions.
5
Identification of 2E7 mAb Target Protein
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For identifying the target protein of mAb 2E7, ARPE-19 cells (10 million) lysed with 0.5% NP40 lysis mix26 (link) were incubated with 2E7 mAb pre-bound to protein G-agarose beads (RepliGen) for 6 h at 4 °C. The recovered proteins were resolved on a SDS-polyacrylamide gel (12.5%) and visualized using Gel Code Blue Reagent (Thermo Fisher). Polypeptides migrating at 60 kDa and 50 kDa were excised and subjected to mass spectrometry proteomic analysis at Bioproximity, Inc (Chantilly, VA)31 (link).
6
Recombinant Tyrp1 and OCA3 Mutants
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Recombinant human wild-type intra-melanosomal domain of the tyrosinase-related protein (residues 25–537 of the native protein), Tyrp1, and OCA3-related mutants, C30R, H215Y, D308N, and R326H, were expressed in baculovirus and produced in whole insect T. ni larvae. Proteins were purified using methods previously described [28 (link),29 (link)], and analyzed by SDS-PAGE using 4–15% polyacrylamide gels (Bio-Rad, Hercules, CA, USA) stained with Thermo Scientific GelCode Blue reagent (Thermo Scientific, Schaumburg, IL, USA). Protein identity was confirmed by Western blot analysis using an anti-Tyrp1 antibody (TA99, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500 dilution).
7
Peptide Conjugation and Characterization
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Synthetic peptides were produced by Mimotopes (Supplementary Tables 6 ,7 ) and Pepscan (Supplementary Table 8 ). Peptide-PMOs were produced by Cambridge Research Biochemicals. With the exception of PAS_SpyC proteins, SpyCatcher conjugations were performed with a SpyC protein: SpyT peptide ratio of 1: 1.25, at a 40 µM final concentration for the SpyC protein or 50 µM final concentration for SpyC_PAP conjugations. Conjugation reactions were incubated for 2 h at 22 °C with gentle mixing and then left overnight at 4 °C. Conjugation efficiencies were analyzed on 4–16% SDS-PAGE gels stained with Gel Code Blue Reagent (Thermo Fisher Scientific). PAS_SpyC proteins were conjugated with SpyTag peptides at a ratio of 1: 1.1, and incubated at room temperature for 30 min before overnight incubation at 4 °C.
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