PhosphoProtein Purification Kit

For purification of phosphorylated proteins by affinity chromatography

Features

  • Complete separation of phosphorylated and unphosphorylated proteins
  • One kit contains columns, buffers, reagents, and ultrafiltration columns
  • Cell-signaling studies without the need for radioactivity

Product Details

The PhosphoProtein Purification Kit, which is based on affinity chromatography, delivers a complete separation of phosphorylated and unphosphorylated proteins from a cell lysate, and therefore facilitates investigation of the phosphorylation status of both entire cells and specific proteins. In addition, the ratio of phosphorylated to unphosphorylated forms of proteins can easily be determined. The drastic reduction of the complexity of each fraction is especially useful when studying proteins of low abundance. The PhosphoProtein Purification Kit includes columns, reagents, buffers, and Nanosep Ultrafiltration Columns for efficient concentration and desalting of protein fractions.

Performance

The PhosphoProtein Purification Kit allows for highly specific separation of phosphorylated proteins and complete separation of unphosphorylated and phosphorylated proteins (see figures  Highly specific separation of phosphorylated proteins and  SDS-PAGE analysis of flow-through and eluate fractions). The PhosphoProtein Purification Kit is also applicable in proteome analysis (see figure  Efficient enrichment for 2D-PAGE phosphoproteome analysis).
See figures

Principle

The PhosphoProtein Purification Kit is based on an affinity chromatography process and provides complete separation of phosphorylated and unphosphorylated proteins from a cell lysate, and therefore facilitates investigation of the phosphorylation status of both entire cells and specific proteins. Phosphorylated proteins can be identified after blotting using highly specific mouse monoclonal PhosphoThreonine and PhosphoSerine Antibodies, which recognize an epitope consisting of either a phosphothreonine or phosphoserine residue, irrespective of the surrounding amino acids. See figure  Complete separation of unphosphorylated and phosphorylated proteins.

See figures

Procedure

Each PhosphoProtein Purification Column can be used to purify phosphorylated proteins from 107 eukaryotic cells (equivalent to approximately 2.5 mg total protein). Cells are lysed in the supplied lysis buffer, which contains a detergent and nucleases to resolve protein complexes, and protease inhibitors to prevent protein degradation. Cleared lysates are loaded onto the PhosphoProtein Purification Column, where phosphorylated proteins in the lysate bind to the column, while unphosphorylated proteins are found in the flow-through fraction. After a wash step, phosphorylated proteins are eluted from the column (see flowchart  Phosphoprotein purification procedure ). Typically approximately 10% of the total protein loaded is in the phosphorylated fraction (depending on cell type and status). Both fractions retain biological activity and can be further purified if desired. Nanosep Ultrafiltration Columns are supplied with the PhosphoProtein Purification Kit to enable efficient concentration and desalting of protein fractions. The table shows typical protein yields in each fraction after separation using the PhosphoProtein Purification Kit.

Yields of phosphorylated proteins obtained using the PhosphoProtein Purification Kit.
Cell type Number
of cells
processed
Total protein in cell lysate (µg) Protein loaded
onto column (µg)
Protein
in eluate (µg)
Percentage
phosphorylated proteins
CHO 1.5 x 107 3400 2500 300 12%
NIH 3T3 n.d. 2750 2500 165 7%
293 1.5 x 107 3650 2500 200 8%
Cos-7 4.5 x 106 1700 1700 120 7%
Huh-7 8.5 x 106 2650 2500 235 9%
HT 29 n.d. n.d. 2500 200 8%
LT 23 n.d. n.d. 2500 275 11%
HeLa S3 1.8 x 107 5950 2500 280 11%
HeLa Acc57 6.6 x 106 2500 2500 235 9%

See figures

Applications

The PhosphoProtein Purification Kit provides complete separation of phosphorylated and unphosphorylated protein fractions for research in:

  • Proteomics 
  • Cell signaling and apoptosis
  • Protein kinases and oncology
  • Immune disorders

Supporting data and figures

Specifications

FeaturesSpecifications
applicationsSDS-PAGE, mass spectrometry
startmaterialCells
fractionsisolatedTwo fractions
proteinwithposttranslationalmodificationPhosphorylation
samplesize~1 x 10e7 cells
speciesEukaryotes
bindingcapacityyield500 µg

Resources

Kit Handbooks (2)
For purification and detection of phosphorylated
proteins from eukaryotic cells and tissues

FAQ

How can air bubbles in PhosphoProtein Purification Columns be avoided?
Air bubbles forming in the columns of the PhosphoProtein Purification Kit can reduce the resin surface area available for protein binding, and thus reduce the binding capacity of the column. To avoid this, it is advisable to degas the PhosphoProtein Lysis Buffer prior to equilibrating the column. Another possibility is to mix the resin with the column storage buffer by inverting the column several times, and let the resin resettle. Always detach the top cap first before opening the bottom closure.
FAQ ID -1070
Can I use the PhosphoProtein Purification Kit to purify phosphorylated peptides of around 1 kD size?

We have not tested the PhosphoProtein Purification Kit with phosphorylated peptides, but we would predict that it should work fine. However, note that the Nanosep Ultrafiltration Columns for concentrating protein fractions will not be suitable due to their cutoff size of 10 kD. The peptides have to be solubilized in, or dialysed against the PhosphoProtein Lysis Buffer before applying the sample to the PhoshoProtein Purification Column.

FAQ ID -778
Which Qproteome or Protein Fractionation Kits from QIAGEN are compatible with tissue samples?

The Qproteome Cell Compartment Kit, the Qproteome Nuclear Protein Kit, the Qproteome Mitochondria Isolation Kit, and the PhosphoProtein Purification Kit are compatible with tissue samples. The respective protocol can be found in the respective handbook.

The Qproteome Mammalian Protein Prep Kit is also compatible with tissue, there is the QIAGEN Supplementary Protocol: Purification of protein from animal tissues using the Qproteome Mammalian Protein Prep Kit and the TissueRuptor™ available. For all these protocols the TissueRuptor is used.

 

 

 

FAQ ID -755
Are HEPES, MOPS and PIPES buffers compatible with QIAGEN's PhosphoProtein Purification kit?

No, these buffers are not recommended for use with the PhosphoProtein Purification Kit. The PhosphoProtein Lysis Buffer supplied in the kit is essential for optimal binding conditions of phosphoproteins to the purification columns. If other lysis buffers must be used, we recommend to dialyse the samples against the PhosphoProtein Lysis Buffer, which can be ordered separately (1000 ml) using catalogue number 1031358.

Alternatively, you can also dilute your lysed sample 1:10 with the PhosphoProtein Lysis Buffer before loading the column.

FAQ ID -482
Do you have any recommendations for using the PosphoProtein Kit with starting material other than cells?

Customers report good results when achieving efficient lysis of their starting material using their home-made lysis buffers, and subsequently dialysing their lysate against the PhosphoProtein Lysis Buffer provided in the PhosphoProtein Purification Kit . Complete exchange of the home-made lysis buffer with the PhosphoProtein Lysis buffer is crucial. The PhosphoProtein Lysis buffer is specifically formulated to bind phosphorylated protein to the column, and is absolutely essential for the procedure.

FAQ ID -597
Can I use the PhosphoProtein Purification Kit for the isolation of phosphorylated proteins from yeast?

The PhosphoProtein Purification Kit is designed for the isolation of phosphoproteins from mammalian cells.

However, there is a customer reference available mentioning the successful enrichment of phosphoproteins from yeast as starting material using this kit, and it may therefore be worth a try. The reference is "Rapid enrichment and analysis of yeast phosphoproteins using affinity chromatography, 2D-PAGE and peptide mass fingerprinting" by Makrantoni et al., Yeast, 2005, 22(5): 401-14.

FAQ ID -838
Is it possible to use the PhosphoProtein Purification Kit in batch format?
We do not recommend to use the resin of the PhosphoProtein Purification Kit in a batch procedure as this will result in reduced yields and unspecific binding of proteins. The columns in the kit are optimized for binding phosphorylated proteins by gravity flow.
FAQ ID -1069
What negative (unphosphorylated) and positive (phosphorylated) control proteins do you recommend for use with the PhosphoProtein Purification Kit?

We have used HSP-60 as a negative control with the PhosphoProtein Purification Kit because it is one of the very few proteins in eukaryotic cells that are always unphosphorylated. Alternatively, delipidated (i.e. phospholipid-free) BSA can be used.

Casein, which is completely phosphorylated, can be used as a positive control. Alternatively, Ovalbumin, which is not completely phosphorylated, may be used. It should bind at around 40-70%.

FAQ ID -779
What are the most commonly used protease inhibitors?

The most commonly used protease inhibitors and their working concentrations are included in the table below:

Protease inhibitor Inhibits Suggested working concentration Stock solution (200x)
PMSF* Serine proteases and cysteine proteases such as papain 85 µg/ml (0.5 mM) 17 mg/ml (200 mM) in ethanol or isopropanol
Leupeptin Serine and thiol proteases 0.5 µg/ml (1 µM) 0.1 mg/ml (200 µM) in water
Pepstatin Aspartic proteases 0.7 µg/ml (1 µM) 0.14 mg/ml (200 µM) in ethanol
Aprotinin Serine proteases 1 µg/ml (0.15 µM) 0.2 mg/ml (30 µM) in water
Pefabloc® Serine proteases 0.5 mg/ml (2 mM) 100 mg/ml (400 mM) in water
Na2-EDTA Metalloproteases 0.35 mg/ml (0.75 mM) 70 mg/ml (150 mM) in water, pH 8.0

* PMSF is inactivated in aqueous solutions and should be added to buffers immediately before use. The half-life of an aqueous solution of PMSF is around 30 minutes at pH 8.

All these protease inhibitors are normally prepared as stock solutions and stored in aliquots at –20°C. Stock solutions are usually stable for up to six months. In addition, ready-to-use mixtures of protease inhibitors are available. Complete Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals) are available in an EDTA-free formulation and are recommended for use during the purification of 6xHis-tagged proteins under native conditions.

FAQ ID -53
Does the His-tag interfere with the purification procedure of the QIAGEN PhosphoProtein Purification Kit?
No, the His-tag does not interfere with the binding of phosphorylated protein to the PhosphoProtein Purification Columns.
FAQ ID -598
Do you provide control proteins for use with the PhosphoProtein Purification Kit?

No, we do not currently provide controls for this kit. BSA can be used as a negative control, since it is not phosphorylated. Use only high-purity grade BSA without any residual phopholipids.

We recommend casein or ovalbumin as a positive control. These proteins can be detected using our PhosphoSerine and PhosphoThreonine Antibody.

FAQ ID -596
Is it possible to use less than 1 mg of protein with the PhosphoProtein Purification Kit?
We do not recommend to use less than a 1 mg of protein with the PhosphoProtein Purification Kit. The technology will work in principle, but the very low-concentration eluate is difficult to handle.
FAQ ID -599
Can I use another lysis buffer than the one supplied in the PhophoProtein Purification Kit?
We strongly recommend to use the lysis buffer supplied with the PhosphoProtein Purification Kit. If your cells cannot be lysed with this buffer, it is principally possible to use your own lysis buffer. In this case, it is crucial for purification that the lysed sample is dialyzed against the PhosphoProtein Lysis Buffer before loading the sample onto the column. Alternatively, you can also dilute your lysed sample 1:10 with the PhosphoProtein Lysis Buffer before loading the column.
FAQ ID -645