Affinity purification, gel-electrophoresis and protein sequencing.
Avi-Link™ columns are produced with the same immobilisation chemistry as the Avi-Chemix™ microarrays. The chemical modification of the surface allows for the immobilisation of a very wide range of compounds. The immobilisation does not require a specific functional group and can be with a wide range of small molecules. The immobilising surface is pre-treated; reacted with special functionalised silane derivatives; washed; dried and further reacted with different types of functional groups. This surface with universal binding capacity can then be used to immobilise the unmodified, low molecular weight, organic molecules.
Total protein extracts are prepared from the cell lysate of choice and are processed with a protease, kinase and phosphatase inhibitor containing buffer. Proteins that bind to the resin are removed from the lysate before it is brought onto the affinity column. The effluent is divided into five fractions that are subsequently concentrated with molecular exclusion membrane tubes.
The concentrated fractions with loading buffers are brought onto acrylamide gels. After SDS polyacrylamide gel-electrophoresis the gels are stained, photographed, destained and the relevant protein bands cut out.
Gel fragments are reduced with DTT, alkylated with iodoacetamide and trypsine digested. After extraction of the peptide fragments, the samples are dried and dissolved in formic acid. The samples are further analysed with a Bruker Reflex III MALDI-TOF mass spectrometer.