Immobilized metal affinity chromatography, or IMAC, has been employed for many applications making use of its versatile interaction with aromatic nitrogens and phosphates. To this end, NTA matrices have been loaded with a broad range of transition metals, as optimized for the individual interaction. Fe-NTA Agarose is often used for the purification of phosphorylated proteins. Alternative matrices are Al-, Ga- or Zr-NTA.
PureCube Fe-NTA Agarose provides:
- Ready-to-use matrix
- Purification of phosphorylated proteins
- Enrichment of iron-binding proteins
- Fe-NTA MagBeads also available
Purification of phosphorylated proteins
IMAC methods, in particular Fe-NTA or Ga-NTA, have been widely used to enrich phosphoproteins and phosphopeptides as part of the sample preparation for mass spectrometry (1,3,4). Depending on the sample, and the kind of phosphorylated proteins to be analyzed, also other transition metals, such as zirconium or aluminium have been loaded on NTA or IDA matrices for enrichment (5). Also, magnetic beads have been useful for this kind of sample preparation (2).
||Specific binding to Iron-binding and Phosphorylated proteins
||Affinity to Iron-binding and Phosphorylated proteins
|Protein Binding Capacity
||The protein binding capacity varies and depends on the specific application.
||pH 2-14, 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% (v/v) acetonitrile.
||Delivered as a 50 % suspension
|A.||Protein purification protocol|
- Prepare: IMAC binding buffer: 40-50% ACN, 0.1 % TFA in H2O
- Wash beads 3 times with 20 volumes of IMAC binding buffer
- Resuspend beads in IMAC binding buffer to make a 50% gel slurry
- Use 100:1 (μg/μL) peptide-to-IMAC ratio: 1 mg of desalted, lyophilized whole-cell digest in 500 μL of IMAC binding buffer and 20 μL of bead slurry
- Incubate for 60 min with vigorous shaking.
- Wash 3 times with 500 μL IMAX binding buffer
- Elute by shaking 5 min in 40 μL of 50 mM K2HPO4/NH4OH, pH 10
- Repeate the previous elution step 3 times to fully recover the phosphopeptides.
- Combine eluates and immediately acidify with 40 μL of 10% formic acid.
1. Albuquerque, C.P. et al. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol Cell Proteomics (2008), 7(7), 1389-1396.
2. Herskowitz, J., et al. Phosphoproteomic analysis reveals site-specific changes in GFAP and NDGR2 phosphorylation in frontotemporal lobar degeneration. J. Proteome Res (2010), 9(12):6368-6379.
3. Yu, P. et al. Global analysis of neuronal phosphoproteome regulation by chondroitin sulfate proteoglycans. PLoS One (2013), 8,3, e59285.
4. Aryal, U.K. et al. Optimization of immobilized Gallium (III) ion affinity chromatography for selective binding and recovery of phosphopeptides from protein digests. Journal of Biomolecular Techniques (2008), 19:296-310.
5. Block et. al. Immobilized-metal affinity chromatography (IMAC) a review. Methods Enzymol. (2009), 463:439-73.