HTRF Human and Mouse Mutant Ataxin 2 Detection Kit, 500 Assay Points

Human mutant Ataxin 2 specification table

Validation of HTRF mutant Ataxin 2 detection kit on HAP1 KO cell line

Three Ataxin 2 plasmids were designed with 3 different repeats of glutamine. 30, 54 or 108 repeats were included in the protein sequence.

HAP1 Ataxin 2 KO cells were cultured upon 80% of confluency in a T175 flask. Transfections of cells took place for 6 hours with plasmids, then the medium was removed, and cells were incubated overnight in culture medium. Lysis buffer was added after the culture medium was removed. The flask was incubated for 30 minutes under gentle shaking.

Lysates were then collected and diluted to be tested with the Ataxin 2 and the Mutant Ataxin 2 detection kits. The Ataxin 2 concentration in the sample was estimated using the HTRF Human and Mouse Ataxin 2 Detection Kit.

As one antibody is specific to the polyQ region, the HTRF signal increased with the number of repeats even if the total concentration of protein was similar.

Plasmid form	30Q	54Q	108Q
[Ataxin2] (ng/mL)	2.95	1.84	2.5

Normal and ALS patient human brain extract test using HTRF total and mutant Ataxin 2 detection kits

Human brain extracts from a normal patient and a patient with ALS were lysed and homogenized using a GentleMACS dissociator following the protocol described in the entitled application note "Best practices for analyzing brain samples with HTRF".

The soluble fractions were collected and the total amounts of protein were defined. The samples (0.125 mg/mL total protein) were then split to follow or not a disaggregation treatment using the Protein Disaggregation kit (part 64DAGGRPEG).

Samples were then tested with the HTRF Total Ataxin 2 and the Mutant Ataxin 2 Detection Kits. 

The Ataxin 2 protein was more highly expressed in the ALS patient sample, with a high aggregation rate. The normal patient sample did not show any aggregation of the protein.

As expected, the mutant form of the protein was only detectable in the ALS patient sample. The disaggregation step resulted in a positive assay signal.