Protein was harvested from zebrafish melanomas 2–6 weeks post-tumor onset by first euthanizing the fish with tricaine methanesulfonate followed by surgical removal of the tumor. Tumors were triturated and lysed in ice-cold RIPA buffer containing a cOmplete protease inhibitor tablet (Roche). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Life Technologies). Samples were run on 10% polyacrylamide gels, transferred, and developed using fluorophore-conjugated antibodies (LI-COR). Antibodies against the following proteins were used: Mitfa (28 (link)); RAF-1 (c-RAF), pMEK1/2 (S217/221), MEK1/2, p44/42 MAPK (ERK1/2), alpha-Tubulin, pAKT S473, AKT, pc-KIT, c-KIT (Cell Signaling 9422, 9154, 8727, 4695, 38735, 4060, 4685, 3391, 3308, respectively); pERK (Sigma m8159); BRAFV600E (Spring Bioscience E19290); total BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively). All quantitative measurements were calculated based on measurements from a LI-COR Odyssey Imaging System and quantified with Image Studio Lite (v 5.0) software. For background subtraction, a median pixel intensity for regions 3 pixels above and below a selected band was calculated and subtracted from the mean pixel intensity of that band.
Fluorophore conjugated antibodies
Manufactured by LI COR
Fluorophore-conjugated antibodies are laboratory reagents that consist of an antibody molecule covalently linked to a fluorescent dye or fluorophore. These conjugates are used to label and detect specific target molecules in various analytical and imaging techniques, such as flow cytometry, immunofluorescence, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw donkey anti rabbit, by LI COR (3 mentions)
P erk, by Merck Group (3 mentions)
Irdye 680rd goat anti mouse, by LI COR (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor tablet, by Roche (3 mentions)
Total braf, by Merck Group (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Nitrocellulose membrane, by LI COR (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
Total mek, by Cell Signaling Technology (1 mentions)
Total erk, by Cell Signaling Technology (1 mentions)
P mek, by Cell Signaling Technology (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Alpha tubulin, by Cell Signaling Technology (1 mentions)
4 protocols using fluorophore conjugated antibodies
1
Zebrafish Melanoma Protein Extraction and Analysis
2
Detection of Native Proteins in Cell Fractions
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For the detection of native proteins in fractionated cell extracts (S3 Fig ), 1% of supernatant and pellet fraction for each time point in germination were loaded on a 12% SDS-polyacrylamide gel. Migration was conducted at 120 V until the dye front reach the bottom of separating gel. Proteins were then transferred to a nitrocellulose membrane (Li-Cor) and blocked for 2 h at room temperature in a blocking Buffer (Li-Cor). The following antibodies were used for detection of Bcy1, Homocitrate synthase and actin, respectively: anti-Bcy1 (yN-19, Santa Cruz Biotechnology, SC-6764), anti-homocitrate synthase (31F5, Santa Cruz Biotechnology, SC-57832), and anti-actin (Clone C4, EMD Milipore, MAB1501R). After washing in phosphate buffered saline (PBS) 1× containing 1% Tween 20, membranes were incubated with the appropriate fluorophore-conjugated antibodies (Li-Cor). Blots were then imaged on an Odyssey Imager (Li-Cor), and images were analyzed on Image Studio software (Li-Cor, v1.1).
3
Western Blot Analysis of Cell Signaling
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Cells were lysed in ice-cold RIPA buffer containing a Complete protease inhibitor tablet (Roche). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Life Technologies). Samples were run on 10% polyacrylamide gels, transferred, and developed using fluorophore-conjugated antibodies (LI-COR). Primary antibodies and dilutions used were: Total BRAF, 1:1000 (10146, Millipore); TetR, 1:1000 (631132, Takara); TUBULIN 1:1000 (2144S, Cell Signaling); BRAFV600E 1:1000 (E19290, Spring Bioscience); RHOA(GTP) 1:500 (ARH03, Cytoskeleton); Total RHOA, 1:500 (ARH05, Cytoskeleton), RAC1 1:500 (ARC03, Cytoskeleton); p-MPS1 T676 1:1000 (PA5-64614, Thermofisher); Total MPS1 (PA5-116969, Thermofisher); p-MEK 1:1000 (9154, Cell Signaling); Total MEK 1:1000 (8727, Cell Signaling); p-ERK 1:1000 (m8159, Sigma); Total ERK 1:1000 (4695, Cell Signaling); P53 1:1000 (sc-126, SCBT); GAPDH 1:5000 (AM4300, Thermofisher). Secondary antibodies and dilutions used were: IRDye 800CW Donkey anti-Rabbit 1:10,000 (LI-COR 926-32213); IRDye 680RD Goat anti-Mouse 1:10,000 (LI-COR 926-68070). Uncropped and unprocessed scans of western blots are provided in Source Data.
4
Western Blot Analysis of MAPK Pathway
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Cells were lysed in ice-cold RIPA buffer containing a Complete protease inhibitor tablet (Roche). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Life Technologies). Samples were run on 10% polyacrylamide gels, transferred, and developed using fluorophore-conjugated antibodies (LI-COR). Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).
Additional information is provided in Extended Experimental Procedures.
Additional information is provided in Extended Experimental Procedures.
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