Western Agarose Native GeELution (WANGEL) with beta-amyloid and tau: Novel method to elute proteins or peptides using native agarose gels followed by Lumipulse assay

Dhwani S Korde; Christian Humpel

doi:10.1016/j.mex.2022.101779

Western Agarose Native GeELution (WANGEL) with beta-amyloid and tau: Novel method to elute proteins or peptides using native agarose gels followed by Lumipulse assay

MethodsX. 2022 Jul 6:9:101779. doi: 10.1016/j.mex.2022.101779. eCollection 2022.

Authors

Dhwani S Korde¹, Christian Humpel¹

Affiliation

¹ Laboratory of Psychiatry and Experimental Alzheimer's Research, Medical University of Innsbruck, Innsbruck 6020, Austria.

Abstract

Alzheimer´s disease is characterized by hyperphosphorylated tau neurofibrillary tangles and beta-amyloid plaques. Both molecules can be easily measured in human fluids or tissue extracts by immunoassays. However, the different molecular weight species can only be differentiated on Western Blot gels. Analysis of native proteins from polyacrylamide gels is also not well characterized. Hence, we developed a modified method to elute proteins or peptides from native agarose gels. Initially, full-length tau (60 kDa) and beta-amyloid(42) (4 kDa) were separated on a Western Blot gel and eluted from native agarose gels (WANGEL) using an elution system inside a polypropylene tube. The eluates were analyzed with the Lumipulse immunoassay. Both molecules were successfully eluted into 1% agarose gels to the cathode and were detected in the eluate. Additionally, tau was eluted from mouse cortical extracts, but was below the detection limit when eluted from human cerebrospinal fluid. Beta-amyloid(40) was eluted from CSF extracts and detected by Lumipulse. In cortical extracts taken from transgenic mice (APP_SweDI) beta-amyloid(42) was detectable as a native peptide and small oligomeric aggregates. Taken together, our novel WANGEL method enables fast, easy and cheap elution of protein/peptides from polyacrylamide/agarose gels with a subsequent analysis by Lumipulse immunoassay. Three bullet points:•Beta-amyloid and tau are major hallmarks in Alzheimer´s disease and are established cerebrospinal fluid biomarkers.•Lumipulse is a method to measure beta-amyloid and tau in cerebrospinal fluid in the pg/mL range.•Western Blot and our novel combined native agarose method (WANGEL) allows an easy and fast determination of the molecular size in combination with Lumipulse.

Keywords: AD, Alzheimers disease; Ab(40), beta-amyloid, 40 amino acids; Ab(42), beta-amyloid, 42 amino acids; Agarose gel; Alzheimer's disease; Beta-amyloid; CJD, Creutzfeld Jakob Disease; CSF, cerebrospinal fluid; Elution; Lumipulse ELISA; PVDF, polyvinylidene fluoride; Tau; WANGEL, Western Agarose Native GeELution; Western blot.