Quantitative Western Blots and Quantitative RT-PCR (qWestern and qPCR) and ELISA
When commercially available, ELISA provides a fast, quantifiable, and convenient approach to monitor the protein level changes for the target of interest.
Scientists at PsychoGenics are able to quickly examine gene expression changes on protein and mRNA levels using Quantitative Western blotting and qRT-PCR. Western blotting can be used to monitor post-translational modifications, and to understand the mechanism of action for drug candidates. Expression level of a gene of interest can be normalized to the means of expression level of three housekeeping genes (qPCR) or three housekeeping proteins (qWestren blot) to provide more accurate quantification.
qPCR assay detected genotype and age dependent decrease of GLT1 expression in the cortex of R6/2 HD mouse model.
Figure 1a. Genotype-dependent decrease of GLT1 mRNA in the cortex of R6/2 transgenic mice (a preclinical model for Huntington’s disease), when mRNA levels of 6 and 12 week-old R6/2 mice were compared to the corresponding wild type littermates. In this experiment normalization was performed using a geometric means of following three housekeeping genes, ATP5B, CanX and Rpl13a.
qWestern assay detected genotype and age dependent decrease of GLT1 expression in the cortex of R6/2 HD mouse model.
Click here for more validation data.
Figure 1b. Relative protein level of GLT1 in the cortex of R6/2 transgenic mice, assessed by qWestern blotting, mirrors the decreases observed at the mRNA level. In this experiment normalization was performed by using a geometric means of following three housekeeping proteins (ATP5B, Eif4a2, and GAPDH)
Currently, protocols for following targets are developed:
- Brain-derived neurotrophic factor (BDNF) and BDNF isoforms (BDNF I, BDNFII, BDNF IV, BDNF VI, and BDNF IX): qPCR
- BDNF qWestern
- Proenkephalin: qPCR and qWestern
- Dopamine D2 receptors: qPCR and qWestern
- kynurenine 3-monooxygenase (KMO): qPCR and qWestern
- The MAP kinase kinase kinase (MLK2): qPCR and qWestern
- Huntingtin (HTT): qPCR and qWestren
- Dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32): qPCR and qWestern
- Phosphodiasterase 10: qPCR
- Glutamate transporter (GLT1): qPCR and qWestern
- Cannabinoid receptor type 1(CB1): qPCR
- c-Jun N-terminal kinases (JNKs) 1, 2, and 3: qPCR and qWestern
- Several inflammation markers such as TNFɑ,IL-1β, IL-6: ELISA and/or qPCR
- Voltage-dependent calcium channel subunit alpha-2/delta-1(CACNA2D1): qPCR and qWestern
- P38 and phosphorylated P38: qWestern
- cAMP response element-binding (CREB) and phospho-CREB: Western
- ERK1/2 and phosphorylated ERK1/2: qWestern
- GFAP: qWestern
