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Technology

Proteopure's proprietary ProteoHook™ technology allows the isolation of all proteins in a biological sample, regardless of the cell type under study or the harshness of the extraction conditions. The key to the technology is a proprietary reagent (ProteoHook) that binds proteins reversibly via primary amino groups on lysine residues and at the N-termini. Proteins are immobilized on a resin via the ProteoHook reagent and then released from the resin after contaminant removal. Unlike precipitation-based methodologies, proteins are maintained in solution at all times.

The use of the product is depicted in Figure 1 below.

Figure 1: Process flow for ProteoHook 2DE k it. Cell lysis and protein solubilization can be carried out as desired. The cell extract is treated with ProteoHook, and then transferred to a spin column containing the ProteoHook resin. All non-protein components are washed out of the column. Additional washing with appropriate buffers and spinning removes all contaminants. Proteins can then be recovered from the column by adding the elution buffer that detaches proteins from the resin in the column. The resulting protein extract is pure for further proteome analysis.

The core ProteoHook technology is exclusively licensed from Carnegie Mellon University. It was invented by Jonathan Minden, Ph.D. Dr. Minden previously invented 2D DIGE (two dimensional differential gel electrophoresis system), now considered one of the most useful tools in comparative proteomics.

Key features of the ProteoHook 2DE Sample Preparation Kit:

• The kit isolates proteins from contaminants without the need for precipitation and resolubilization.

• It eliminates 95% or more of salts, nucleic acids, lipids and detergents including SDS (Figure 2). Thus aggressive methods, such as boiling in SDS, may be used for more complete harvesting of proteins from cells.

• It maintains relative ratios of the various proteins in a complex mixture thereby improving reliability and reproducibility of results (Figure 3).

• The kit can be used with protein samples from any source. Having a standardized protocol decreases the time spent optimizing sample preparation and protein isolation protocols.

Figure 2: Ability of the ProteoHook 2DE kit to remove lipids, DNA and SDS from protein samples. BSA samples with a concentration of 1mg/ml were spiked with 14C phosphatidyl choline, 3H DNA or 14C SDS. Radioactivity present in the first three eluates was summed to determine the total % of contaminant left in the sample after treatment with the kit, and thereby the total % removal. Numbers shown are the average of two experiments. In all cases, standard deviation between the two readings was less than 0.5%.

Figure 3: Ability of the ProteoHook 2DE kit to maintain relative protein ratios. A 2D-DIGE experiment was done to compare protein profiles of Drosophila embryo extracts with and without treatment with the ProteoHook 2DE kit. An overlay of the Cy3 and Cy5 images from the same 2DE gel, representing untreated and treated samples respectively, is shown. The image of the untreated sample is pseudocolored green and that of the treated sample is pseudocolored red. Unchanging protein spots appear yellow. Proteins that are present in greater amounts in the untreated sample appear green, while those present in greater amounts in the treated sample appear red. The acidic end of the gel is to the left, basic to the right. Based on analysis of this gel and a duplicate with the labeling scheme reversed, a total of 9 protein changes were observed between untreated and treated samples. Considering that the 2DE gel has approximately 1500 protein spots, this accounts for approximately 0.06% of all proteins on the gel. All 9 differences were small differences in abundance rather than complete disappearance/appearance of a protein.

The ProteoHook 2DE Kit has been tested for compatibility with different cell types. Comparisons with precipitation-based kits have been done (Figure 4). More proteins were found to enter the 2DE gels and protein spot resolution was better when samples were treated with the ProteoHook kit. The kit also performed better than precipitation-based kits at removing SDS from samples prepared in this detergent.
 

Figure 4: Comparison of performance of ProteoHook 2DE kit with a precipitation-based kit. E.coli whole cell extracts and HeLa whole cell extracts were labeled with Cy3 and treated with the ProteoHook 2DE kit or a precipitation-based kit. Drosophila embryo extract made in buffer containing 1% SDS was labeled with Cy3 and treated with the ProteoHook 2DE kit or a precipitation-based kit. Resulting samples were subjected to 2DE. Fluorescence image is shown in each case. In all cases, the acidic end of the gel is to the left and the basic end to the right.

Customer Supplied 2D Gel Images

E.coli cells in 6% SDS (provided by Lee Lab, Cornell)

Serum (after removal of high abundance proteins, Cy3-labeled)

Mammalian Cell Tissue (Cy5-labeled)

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