Phospho-LRRK2 (Ser935) & Total LRRK2 Cellular Assay Kits

Phospho- and total LRRK2 assays to study Parkinson’s disease

LRRK2 is a significant target in neuroscience research involving Parkinson’s disease (PD) and neurodegenerative disorders in general.

LRRK2 mutations have been linked to the genetic development of Parkison’s. LRRK2 has also been involved in the phosphorylation of Tau protein, leading to its aggregation and onset of dementia.

Cisbio provides an accurate solution to investigate the cellular LRRK2 state of phosphorylation with our cell-based phospho LRRK2 (Ser935) assay combined with Total LRRK2 assay used to normalize against steady-state expression.

These assays are part of our CNS range including Tau phosphorylation and Tau aggregation assays.

Explore LRRK2 Phosphorylation

Explore LRRK2 Phosphorylation with Cisbio

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

The LRRK2 phosphorylation immunoassay is based on an HTRF sandwich format with specific antibodies bound to either donor (cryptate) or acceptor (d2) fluorophores, which enable energy transfer when they bind to the same target protein.

One antibody is directed against the phosphorylated site (Ser 935), the other against LRRK2 protein. When LRRK2 is phosphorylated, both bind to the protein and HTRF can occur.

The Total LRRK2 assay is used to detect both phosphorylated and unphosphorylated forms of LRRK2, and thus acts as a normalization assay against steady-state expression.

It can be run in one plate or with a transfer step where cells are cultivated and treated in the desired format (6 wells, 96 wells …) then lysed and transferred to a 384-well plate for the detection step.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Product performance

Phospho LRRK2 detection in NIH 3T3 cells

Cells were cultured in 6-well plates and treated with increasing concentrations of CZC… and GSK… for 2 hours. Medium was removed and replaced with Cisbio lysis buffer for 1 hour. 16µl of lysate were taken from each well and transferred to a 384-well plate for detection with HTRF reagents.

The Cisbio phosphorylated LRRK2 assay enabled clear determination of both inhibitors’ IC50s, in agreement with literature.

HTRF assay compared to Western Blot using phospho LRRK2 and total-LRRK2 cellular assays

Murine NIH-3T3  embryonic fibroblasts were grown in a T175 flask in complete culture medium and incubated for 2 days at 37°C, 5% CO2 until reaching 100% confluency

After a PBS washing step, cells were lysed with 3 mL of supplemented lysis buffer # 4 for 30 min at room temperature. Soluble fractions were then collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western Blot and by HTRF.

Results show that HTRF Phospho and total LRRK2 cellular assays are more sensitive than the Western Blot:

The HTRF phospho-assay is 8-fold more sensitive than the Western Blot: only 2,500 cells are sufficient for minimal signal detection when using the HTRF phospho- LRRK2 assay while 20,000 cells are needed  for a Western Blot signal.

The HTRF total assay is 2-fold more sensitive than the Western Blot: only 10,000 cells are sufficient for minimal signal detection when using the HTRF phospho- LRRK2 assay while 20,000 cells are needed  for a Western Blot

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
LRRK2 phospho-S935 kit - 500 tests6FLRKPEG
LRRK2 phospho-S935 kit - 10,000 tests6FLRKPEH
LRRK2 phospho-S935 kit - 50,000 tests6FLRKPEY
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LRRK2 total kit - 500 tests 6FNRKPEG
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LRRK2 total kit - 10,000 tests6FNRKPEH
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Companion products

DescriptionCat. noProduct insertMSDS
Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #4 - 130 mL64KL4FDF
LRRK2 phospho-S935 kit control lysate6FLRKTDA
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LRRK2 total kit - control lysate6FNRKTDA
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Documents