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 sample. 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.

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

Anti-Phospho-Tyrosine (pTyr) 2D Western Blot: Jurkat cell proteome 2D gel image

B. Anti-pTyr Western Blot

Anti-Phospho-Tyrosine (pTyr) 2D Western Blot: Anti-pTyr 2D Western Blot image

C. Total Protein / Anti-pTyr

Anti-Phospho-Tyrosine (pTyr) 2D Western Blot: overlay image of Jurkat cell proteome and anti-pTyr 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

Anti-Phospho-Serine (pSer) 2D Western Blot: Hela cell proteome 2D gel image

B. Anti-pSer Western Blot

Anti-Phospho-Serine (pSer) 2D Western Blot: Anti-pSer 2D Western Blot image

C. Total Protein / Anti-pSer

Anti-Phospho-Serine (pSer) 2D Western Blot: overlay image of Hela cell proteome and Anti-pSer 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

Anti-Phospho-Serine/Threonine/Tyrosine (pSer/pThr/pTyr) 2D Western Blot: Hela cell proteome 2D gel image

B. Anti-pSer/pThr/pTyr Western

Anti-Phospho-Serine/Threonine/Tyrosine (pSer/pThr/pTyr) 2D Western Blot: Anti-pSer/pThr/pTyr 2D Western Blot image

C.  Protein / Anti-pSer/pThr/pTyr

Anti-Phospho-Serine/Threonine/Tyrosine (pSer/pThr/pTyr) 2D Western Blot: overlay image of Hela cell proteome and Anti-pSer/pThr/pTyr Western

Sample Info

Please follow these general principles regarding biohazardous materials and buffer condition in getting your samples ready.

Protein amount for each WB: 100-500 µg of total protein from each sample.

Protein concentration: 5-20 mg/ml is preferred.

Primary antibody requirement for each WB: Sufficient for 100ml incubation volume, and need information on:

  • Animal species in which the antibody was raised
  • Suggested dilution factor

If you cannot provide the dilution factor, please let us know:

  • If it is a commercial antibody, provide the link to the antibody information
  • If it is not a commercial antibody, please provide the protein concentration of the antibody, whether it is IgG fraction or anti-sera or antigen-affinity purified.

Buffer: Please feel free to submit samples in whichever buffer that you would normally use. The standard sample preparation is covered by the DIGE gel cost. Such samples should have protein concentration in the range of 5-20 mg/ml. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.

Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.

Pricing

Services DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
CodePriceCodePrice
2D Gel with 1 CyDye Labeling 1401A$800401N$960
2D DIGE Gel with 2 CyDye Labeling 1101A2$1200101N21500
Transfer to PVDF / Nitrocellulose Membrane 2520A$250520N$295
Western Blot 1-color 3512A$410512N$495
Western Blot 2-color 3512ACall for price513NCall for price

Primary antibody: Customers are responsible for providing the primary antibodies. Applied Biomics will provide the following primary antibodies at $150-250 for each Western blot:

  • Anti-Bacteria HCP antibody: E. coli; Lactococcus lactis
  • Anti-Mammalian Cell HCP antibody: Vero Cell; CHO; BHK; HEK 293; A549; MRC5; PER.C6
  • Anti-Yeast HCP antibody: Saccharomyces cerevisiae; Pichia pastoris
  • Anti-Insect Cell HCP antibody: SF9; SF22
  • Others: Anti-Bovine Serum Protein antibody; Anti-Mycobacterium Protein antibody; Anti-Goat-Milk Protein antibody

Fluorescent labeled secondary antibody: Provided by Applied Biomics and included in the price.

1. Price covers:

  • Experimental design
  • Standard sample preparation and protein concentration determination
  • Fluorescent dye labeling using CyDye
  • IEF and SDS-PAGE
  • Gel image scan using Typhoon scanner
  • Image analysis (ImageQuant)
  • Data Report

2. Price covers:

  • Trans-blotting 2D Gel to PVDF or Nitrocellulose membrane
  • Membrane images scanned after the transfer

3. Price covers:

  • Western blot using the primary antibodies provided by customers, Applied Biomics will provide secondary antibodies
  • Membrane images scanned after Western Blot

1-3. Each Western blot will cover 2 hour data analysis generating Protein and Western blot image report.

Price does NOT cover:

  • Primary antibody provided by customer
  • Manual matching spots and manual circling and numbering spots on power point images
  • Customized data request in addition to the standard request