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.
Total braf
Manufactured by Merck Group
Total BRAF is a laboratory equipment product that measures the total amount of the BRAF protein in a sample. BRAF is a protein involved in the regulation of cell growth and division. The Total BRAF product provides a quantitative assessment of the BRAF protein levels, but does not include any interpretation or intended use for the measurement.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor tablet, by Roche (2 mentions)
Irdye 680rd goat anti mouse, by LI COR (2 mentions)
Irdye 800cw donkey anti rabbit, by LI COR (2 mentions)
P erk, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fluorophore conjugated antibodies, by LI COR (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Total erk, by Cell Signaling Technology (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Total mek, by Cell Signaling Technology (1 mentions)
P mek, by Cell Signaling Technology (1 mentions)
2 protocols using total braf
1
Zebrafish Melanoma Protein Extraction and Analysis
2
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!