Home Abstract Introduction
Immunofluorescence of VAPs and Calreticulin Time Course Immunofluorescence of VSVG with relationship to the FFAT motif Immunofluorescence of Calreticulin and Chromogranin B Time Course Immunofluorescence of properly folded VSVG Quantitative Western Immunoblot of VSVG for ER budding Western Blots and Quantification of Microtubule binding of FFAT and AAAT
Implications Support

Translation of Journal of Cell Science

Prosser, D. C., et al.
"FFAT Rescues VAPA-Mediated Inhibition of ER-to-Golgi
Transport and VAPB-Mediated ER Aggregation."
Journal of Cell Science, vol. 121, no. 18, 19 June 2008, pp. 3052-3061.,
doi:10.1242/jcs.028696.

(Translated by Sohail Syed)

Western Immunoblot and Quantification of Microtubule binding of FFAT and AAAT

Motivation

The motivation of this experiment was to show how VAPs react with microtubules. Because the protein complex, VAPs are located in, work with microtubules it is necessary to see if there are any harmful interactions due to the over-expression of VAPs as there are VAPs at contact sites with microtubules, which could affect proteins reaching ER exit sites.

Procedure

Steps for Western Immunoblot and Quantification of Microtubule binding

    1. Prepare microtubules

    2. Steps from Western Immunoblot

    3. Spin microtubules with FFAT and AAAT

    4. Look for FFAT and AAAT association through western blots

Results

If microtubules in pellet there is expression in western blot

FFAT lowers association with microtubules vs AAAT

Because of aggregates there is no need for microtubules