PPP1CA and SMP complex phosphatase activity was tested on purified RAF1 and BRAF proteins. RAF1 and BRAF were diluted in 20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM TCEP, 2 mM MgCl2, 2 mM MnCl2 to a final concentration of 686 nM. 20 ul of diluted RAF1/BRAF sample was mixed with 20 ul of 204 nM SMP or PPP1CA and incubated at 30°C for 30 minutes. After 30 minutes, 40 ul of 2x NuPAGE LDS Sample Buffer (Thermo Scientific, Waltham, MA) was added to the tube and samples were boiled for 5 minutes to stop the reaction. Western blots were prepared by electrophoresing 10 ul of each sample on an SDS-PAGE gel, transferring samples to a PVDF membrane via iBlot (Thermo Scientific, Waltham MA) using standard manufacturer’s conditions, and probing for RAF1 pS259/BRAF pS365 (Abcam #ab173539), RAF1 pS621/BRAF pS729 (Abcam #ab4767), and anti-His6 to detect tagged BRAF and RAF1 proteins (Abcam #ab18184). Final images were taken using an Odyssey CLx (LI-COR Biosciences, Lincoln NE).
Ab4767
Manufactured by Abcam
Ab4767 is a laboratory equipment product. It serves a core function, but its intended use is not provided in this description.
Automatically generated - may contain errors
Lab products found in correlation
Glut1, by Proteintech (2 mentions)
Ab200653, by Abcam (2 mentions)
Ab96379, by Abcam (2 mentions)
Ab17942, by Abcam (2 mentions)
Ab223500, by Abcam (2 mentions)
Gapdh, by Proteintech (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Ab18184, by Abcam (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Ab173539, by Abcam (1 mentions)
3 protocols using ab4767
1
Phosphatase activity on RAF proteins
2
EGFR Signaling Pathway Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Western blots were performed as we protocol described and the protein extracted and detected materials were purchase from Beyotime. The antibodies used in this study were as follows: EGFR (Proteintech, 66455-1-Ig), GLUT1 (Proteintech, 21829-1-AP), HK2 (Proteintech, 66974-1-Ig), PKM (Proteintech, 10078-2-AP), MYC (Proteintech, 10828-1-AP), LDHA (Proteintech, 19987-1-AP), GAPDH (Proteintech, 60004-1-Ig), RAF (Abcam, ab200653), MEK (Abcam, ab96379), ERK (Abcam, ab17942), phosphorylated RAF (Abcam, ab4767), phosphorylated MEK (Abcam, ab96379), and phosphorylated ERK (Abcam, ab223500). Cycloheximide (CST, #2112) was used to evaluate the protein stability of EGFR.
3
EGFR Signaling Pathway Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots were performed as we protocol described and the protein extracted and detected materials were purchase from Beyotime. The antibodies used in this study were as follows: EGFR (Proteintech, 66455-1-Ig), GLUT1 (Proteintech, 21829-1-AP), HK2 (Proteintech, 66974-1-Ig), PKM (Proteintech, 10078-2-AP), MYC (Proteintech, 10828-1-AP), LDHA (Proteintech, 19987-1-AP), GAPDH (Proteintech, 60004-1-Ig), RAF (Abcam, ab200653), MEK (Abcam, ab96379), ERK (Abcam, ab17942), phosphorylated RAF (Abcam, ab4767), phosphorylated MEK (Abcam, ab96379), and phosphorylated ERK (Abcam, ab223500). Cycloheximide (CST, #2112) was used to evaluate the protein stability of EGFR.
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