Protein Disaggregation Kit Principle

The disaggregation principle is based on two successive reagent dispensing steps, without the need for centrifugation. Disaggregation buffer A is added to the sample and left for 15 min at room temperature, then Disaggregation buffer B is added. The same sample is also treated by Control detection buffer C to generate an appropriate control. After treatment, samples are ready to use in immunoassays. Signals from samples containing protein aggregates (treated with Control buffer C) can be compared to the same samples treated with Disaggregation reagents A and B. The ratio obtained between disaggregated/ aggregated signals on the same samples is proportional to level of protein aggregation.

Protein Disaggregation Kit on 1-42 beta amyloid recombinant proteins

Briefly, human beta amyloid 1-42 peptide was diluted in a final volume of 600 µL, in low binding microtubes, at a concentration of 30 µM in a 10 mM sodium phosphate pH 7.4 buffer. The microtube was incubated at 25°C without agitation. Samples were withdrawn at several timepoints, diluted down to 325 ng/mL and flash-frozen in liquid nitrogen prior to their storage at -80°C. For Thioflavin T analysis 20 µL of samples were mixed with 6 µL of 100 µM Thioflavin T solution and 14 µL glycine buffer pH 8.3.

After a 15 min incubation in a 96w half area black clear bottom polystyrene plate, Thioflavin T fluorescence was read at 485 nm with an excitation at 444 nm (reported as blue bars in the figure). For HTRF analysis, samples were 1:100 diluted in Diluent 5 and treated using the Disaggregation Reagents prior to analysis with the human beta amyloid 1-42 HTRF kit (#62B42PEG). HTRF results are analyzed according to the Disaggregation Reagent protocol to compute a Disaggregation Ratio (reported as red bars in the figure).

Protein Disaggregation Kit on 1-42 beta amyloid proteins from nematodes

Briefly, 5000 C. elegans nematodes were prepared for each condition to be tested (stimulated during 24h at 25°C and non-stimulated samples). Prior to analysis, nematodes were washed and centrifuged to form a pellet. Pellets and supernatants were carefully separated, and respective samples were shock-frozen in liquid nitrogen and stored at -80°C. Pellets were suspended with 500 µL of 1X Lysis Buffer 1 supplemented with protease inhibitors and sonicated on ice for 20 sec in 1.5 mL microtubes. Samples were then aliquoted and stored at -80°C until analysis using the human amyloid beta 1-42 HTRF kit and the standard curve being generated in the HTRF Lysis Buffer #1.

Protein Disaggregation Kit on recombinant alpha-synuclein

Recombinant WT (monomer) and A53T (fibrilar mutant, linked to Parkinson’s disease) alpha-synuclein proteins were diluted at a concentration of 100 ng/mL. Samples were processed by the Protein Disaggregation Kit reagents according to the protocol using 60 µL sample and sequential additions of 10 µL of each Disaggregation buffers A then B or 2x10 µL of Detection Control buffer C. Processed samples were then used in the HTRF alpha-synuclein aggregation kit with an overnight incubation according to the kit package insert. HTRF ratios of samples processed using the Control buffer C displays the expected pattern underlining an higher HTRF ratio for the A53T fibrilar mutant, which is expected for the alpha-synuclein aggregation kit which is aimed to detect aggregated alpha-synucleins only. Upon treatment with Disgaggregation buffers A and B, HTRF ratios are similar for both the monomeric and fibrilar proteins, clearly demonstrating that the disaggregation reagents are effective in disaggregating the fibrilar A53T protein into a monomeric form that is not anymore detactable by the alpha-synuclein aggregation kit.

Protein Disaggregation Kit on detection of TDP-43 aggregates

Human HeLa cells (100,000 cells/well) were transfected in a 96 well plate with 200 ng/well of GFP-TDP-43 Wild Type (full length) or GFP-TDP-43 mutated (C Terminal fragment 220-414 aa) using lipofectamine 2000. After 48h incubation, cells were stimulated for 6h with 1 µM of staurosporine (a well-established caspase activator cleaving TDP-43 to induce aggregation of C-terminal fragments). Cells were lysed with 50 µL of supplemented lysis buffer #1. Three wells of the same condition were pooled and 2x60 µL of ¼ diluted lysate were transferred into a 96 well plate prior to the disaggregation or control detection step. After treatment, 16 µL of each sample (disaggregated or control detection condition) were then transferred into a 384 well low volume white microplate before the addition of 4 µL of the HTRF TDP-43 detection reagents. The HTRF signal was recorded after an overnight incubation.

The overexpression of WT or mutated TDP-43 are expected to increase the TDP-43 levels and these effects are only clearly visible after treatment of the samples with the disaggregation buffers A+B. The control condition (buffer C, not disaggregated) shows low signals and unsignificant changes between tested conditions, the epitopes being masked in high-order aggregated structures. Upon disaggregation, basal TDP-43 levels are better detected and effect of transfections are significantly different versus untransfected cells and also between WT and mutant TDP-43 forms. Aggregated ratios are computed according to the kit instructions and are reported in red in the figure. This result clearly demonstrates the critical importance of use of disaggregation reagents in the study of proteins prone to form highly aggregated structures, generating unbiased and more meaninigful biological information.