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PhosphoProtein 2D Western Blot: Detect & Quantify

 

 

 

Our PhosphoProtein 2D Western Blot platform offers high sensitivity detection and accurate quantitation of protein phosphorylation in any protein samples. Customers can choose to detect and quantitate a specific type of Protein Phosphorylation such as pSer or pThr or pTyr. In addition, we offer mass spectrometry service to identify the Protein Phosphorylation sites. Applied Biomics’ 2D Fluorescent Western Blot has been successfully applied in studying Kinase/Phosphatase substrate and other functions of Phospho-Proteins.

Representative PTM 2D Western Blots:

1. Phosphorylation 6. Nitrotyrosine
2. Ubiquitination 7. Biotinylation
3. Acetylated-Lysine 8. Succinyl-Lysine
4. Sulfo-Tyrosine 9. Citrullination
5. Methyl-Arginine 10. 4-hydroxynonenal (4-HNE)



1. Phosphorylation

Example 1. Anti-Phospho-Tyrosine (pTyr) Western Blot

Jurkat cells were treated with serum starvation for 1 hour. Then serum was added to the medium and cultured for 4 hours. Proteins were extracted by 2D lysis buffer following Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). Second, proteins 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. Cell Lysate Total Protein

Phospho-2D Western Blot

B. Anti-pTyr Western Blot

Phospho-2D Western Blot

C. Total Protein / Anti-pTyr Western

Phospho-2D Western Blot

 

Example 2. Anti-Phospho-Serine (pSer) Western Blot

Hela cells were treated with 1 mM sodium orthovanadate for 30 minutes. Proteins were extracted by 2D lysis buffer following Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). 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 (B).

A. Cell Lysate Total Protein

Phospho-2D Western Blot

B. Anti-pSer Western Blot

Phospho-2D Western Blot

C. Total Protein /Anti-pSer Western

Phospho-2D Western Blot

 

Example 3. Anti-Phospho-Serine/Threonine/Tyrosine (pSer/pThr/pTyr) Western Blot

Hela cells were treated with 1 mM sodium orthovanadate for 30 minutes. Proteins were extracted by 2D lysis buffer following Applied Biomics protocols. First, sample was run on 2D DIGE (pH4-7) and the gel image was scanned using Typhoon 9400 (A). Western Blot was performed using mouse monoclonal antibodies against pSer/pTyr/pThr (Sigma) and CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California). The Western Blot image was scanned using Typhoon 9400 (B).

A. Cell Lysate Total Protein

Phospho-2D Western Blot

B. Anti-pSer/pThr/pTyr Western Blot

Phospho-2D Western Blot

C. Total Protein / Anti-pSer/pThr/pTyr

Phospho-2D Western Blot

If you are interested, please follow these steps to obtain more information and order this service.