Improving Protein Array Performance: Focus on Washing and Storage Conditions

Abstract

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.

HaloTag, a Platform Technology for Protein Analysis

“…Single point, oriented attachment increases capacity, effectiveness and reproducibility of the system preventing surface induced protein unfolding and improving protein activity and native conformation [55]. The need for protein specific optimization of capture conditions is also eliminated as the immobilization relies on the same chemistry, namely attachment of HaloTag to the HaloTag ligand-modified surface.…”

The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

“…Independent methods are needed to validate and discover protein-protein interactions [12]. We have used the HaloTag technology in a number of formats as a means of identifying or validating a number of binary protein interactions and also to identify constituents of multi-protein complexes [7, 40]. …”

Cell-Free Expression of Protein Kinase a for Rapid Activity Assays

“…The HaloTag ® Technology was chosen for this purpose because it enables rapid, specific, covalent and oriented binding of HaloTag fusion protein to solid surfaces 8,9. The HaloTag protein is a genetically engineered version of a hydrolase enzyme.…”

“…The kinase was active and inhibited by known inhibitors. cPKA-HaloTag fusion protein expressed in wheat germ extracts has previously been shown to be functional 9. In this instance the kinase was assayed after covalent capture onto glass slides that had been activated with ligand specific for the HaloTag moiety 9.…”