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PhosphoProteomics & PhosphoProtein Array Services

 

 

 

Applied Biomics’ Phospho-Proteomics & PhosphoProtein Array platform offers high sensitivity detection and accurate quantitation of protein Phosphorylation change. Furthermore, customers can choose to target all 3 types of phosphorylation (pSer/pThr/pTyr) by 2D DIGE PhosphoProtein Array, or a specific type by 2D Phospho Western Blot.

Main applications:

- Kinase/Phosphatase substrate mapping
- Kinase/Phosphatase activator screening and identification (such as drug induced protein phosphorylation)
- Kinase/Phosphatase inhibitor screening and identification
- Signal transduction studies (regulation of protein functions by phosphorylation)

Here are some highlighted features of our Phospho-proteomics platform:

- Phospho-protein enrichment (optional)
- Effective separation of phospho-proteins from their unmodified form by high resolution large format 2D gel
- High sensitivity detection and quantitation of phospho-proteins by fluorescent labeling
- Accurate quantitation of changes in protein phosphorylation between different samples
- Identification of phosphorylation sites is available as a follow up experiment (optional)

Based on your project objective, choose one of the following study designs:

A. Identify proteins differentially phosphorylated between 2 or more samples (including all 3 types: Phospho-Ser, Thr & Tyr)

B. Identify proteins differentially phosphorylated at specific amino acid between 2 or more samples by 2D Western Blot

C. Identify proteins that are phosphorylated

 

A. Identify proteins differentially phosphorylated between 2 or more samples (including all 3 types: Phospho-Ser, Thr & Tyr)

*Study design for 2 samples (Control, Test)

Gel-1: Control, Phospho-profiling
Gel-2: Test, Phospho-profiling
Gel-3: Control, Test

Image reports:

- Gel-1: Total Protein image, PhosphoProtein-image and overlay image of Total Protein/PhosphoProtein
- Gel-2: Total Protein image, PhosphoProtein-image and overlay image of Total Protein/PhosphoProtein
- Gel-3: Protein image of each sample and overlay of the 2 samples

Gel-1

Control: Total Protein

2D DIGE In-Gel Data Analysis

Control: PhosphoProtein

2D DIGE In-Gel Data Analysis

Total Protein / PhosphoProtein

2D DIGE In-Gel Data Analysis

Gel-2

Test: Total Protein

2D DIGE In-Gel Data Analysis

Test: PhosphoProtein

2D DIGE In-Gel Data Analysis

Total Protein / PhosphoProtein

2D DIGE In-Gel Data Analysis

Gel-3

Control

2D DIGE In-Gel Data Analysis

Test

2D DIGE In-Gel Data Analysis

Control / Test

2D DIGE In-Gel Data Analysis

Quantitation report:

- Phospho-ratio: Gel-1 & 2 will provide phospho-signal of each phosphorylated spots and phospho-ratio between 2 samples
- Protein ratio: Gel-3 will provide the protein ratios
- Final Phospho-ratio: Calculated by adjusting Phospho-ratios with protein ratios

2D DIGE In-Gel Data Analysis

 


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B. Identify protein differentially phosphorylated at specific amino acid between 2 or more samples by 2D Western Blot

*Study design for 2 samples

Gel-1: Control, Western Blot with anti-pSer or pThr or pTyr antibody
Gel-2: Test, Western Blot with anti-pSer or pThr or pTyr antibody
Gel-3: Control, Test

Image reports:

- Gel-1: Total Protein image, PhosphoProtein WB image and overlay image of Total Protein/PhosphoProtein
- Gel-2: Total Protein image, PhosphoProtein WB image and overlay image of Total Protein/PhosphoProtein
- Gel-3: Protein image of each sample and overlay of the 2 samples

Quantitation report:

- Phospho-ratio: Gel-1 & 2 will provide phospho-signal of each phosphorylated spots and phospho-ratio between 2 samples
- Protein ratio: Gel-3 will provide the protein ratios
- Final Phospho-ratio: Calculated by adjusting Phospho-ratios with protein ratios

*Examples of 2D Western Blot with Anti-pSer or Anti-Tyr

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

2D DIGE In-Gel Data Analysis

B. Anti-pTyr Western Blot

2D DIGE In-Gel Data Analysis

C. Total Protein / Anti-pTyr Western

2D DIGE In-Gel Data Analysis

 

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

2D DIGE In-Gel Data Analysis

B. Anti-pSer Western Blot

2D DIGE In-Gel Data Analysis

C. Total Protein / Anti-pSer Western

2D DIGE In-Gel Data Analysis

 

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

2D DIGE In-Gel Data Analysis

B. Anti-pSer/pThr/pTyr Western Blot

2D DIGE In-Gel Data Analysis

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

2D DIGE In-Gel Data Analysis


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C. Identify proteins that are phosphorylated

*Study design for 1 sample

Gel-1: Cell lysate, Phospho-profiling or Phospho-WB

Cell Lysate Total Protein

2D DIGE In-Gel Data Analysis

PhosphoProtein

2D DIGE In-Gel Data Analysis

Total Protein / PhosphoProtein

2D DIGE In-Gel Data Analysis

*Study design for 2 samples:

The phospho-signal is the combined effects of both samples. If you want to obtain phospho-signal for each sample, please use Study design A.

Gel-1: Normal, Drug treated, Phospho-profiling or Phospho-WB

Image report (for 2 samples):

- Gel-1: Protein image of each sample and overlay of the 2 samples; Phospho-image of 2 samples and overlay image of Protein/Phospho

Green: Normal

Phosphorylated-Protein Expression Profiling
Red: Drug treated

Phosphorylated Protein Expression Profiling

Green: Normal / Red: Drug treated

Phosphoprotein

Blue: PhosphoProtein Array

Phospho-protein


Green: Normal / Blue: PhosphoProtein Array

Phosphorylated-Protein Expression Profiling
Normal / Treated / PhosphoProtein Array

Phosphoprotein Array

 

Quantitation report:

- Protein ratios will be provided on differently expressed protein spots
- Phopsho-spots will be marked on the protein ratio table


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