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Home > Proteomics Services > 2D Phosphorylated-Western Blot
2D Phosphorylated-Western Blot
Figure 1.
Jurkat cells were treated with serum starvation for 1 hour followed by adding serum to the culture medium, and culture for 4 hours before harvesting the cells. Protein extraction was prepared using 2D lysis buffer using Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). Second, proteins from the 2D gel were transferred to the membrane and Western Blot was performed using mouse monoclonal anti-phosphotyrosine antibody (Clone PY20) and CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California). The Western Blot image was scanned using Typhoon 9400 (B)
A. Gel image captured before the transfer
B. Fluorescent 2D Western Blot using anti-pTyr

phospho_westernblot image
Fluorescent 2-D Western Blot
Figure 2.
Hela cells were treated with 1 mM sodium orthovanadate for 30 minutes, and then protein extraction was prepared using 2D lysis buffer using Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (C). Second, proteins from the 2D gel were transferred to the membrane and the membrane image is captured (E). Western Blot was performed using mouse monoclonal anti-phosphoserine antibody (Sigma) and CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California). The Western Blot image was scanned using Typhoon 9400 (D and F).
C. Gel image captured before the transfer
D. Fluorescent 2D Western Blot using anti-pSer
phospho_westernblot image
Fluorescent 2-D Western Blot

E. Protein image captured from membrane

F. 2D Western blot using anti-pSer(black and white)
Protein image captured from membrance
2D Western Blot
Figure 3.
Hela cells were treated with 1 mM sodium orthovanadate for 30 minutes, and then protein extraction was prepared using 2D lysis buffer using Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (G). Second, proteins from the 2D gel were transferred to the membrane and Western Blot was performed using pooled mouse monoclonal antibodies against phosphotyrosine, phosphoserine and phosphothreonine. The Western Blot image was scanned using Typhoon 9400 (H)
G. Gel image captured before the transfer
H. Western Blot: anti-pSer, anti-pTyr and anti-pThr
phospho_westernblot image
Western Blot

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