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Phosphorylation and Other Post-Translational Modifications (PTM) 2D Western Blot

 

 

 

1. Phospho-2D Western Blot
2. Ubiquitination
3. Acetylated-Lysine
4. Sulfo-Tyrosine
5. Methyl-Arginine
6. Nitrotyrosine

7. Biotinylation

1. Phospho-2D Western Blot

Example 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

Phospho-2D Western Blot 
B. Fluorescent 2D Western Blot using anti-pTyr

Fluorescent 2D Western blot using anti-pTyr

 

Example 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

Phospho-2D Western Blot
D. Fluorescent 2D Western Blot using anti-pSer

Fluorescent 2D Western blot using anti-pSer

E. Protein image captured from membrane

Phospho-2D Western Blot

F. 2D Western blot using anti-pSer(black and white)

2D Western blot using anti-pSer

 

Example 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

Phospho-2D Western Blot
H. Western Blot: anti-pSer, anti-pTyr and anti-pThr

Western blot:anti-pSer, anti-pTyr and anti-pThr