Motivation

The motivation for this experiment was to find the differences between FFAT motif and AAAT motif. This experiment calls for looking for expression of VSVG budding as it implies efficiency of the cell in transmitting proteins into the vesicles.

Procedure

Steps for preparing Western Immunoblot data:

1. Prepare cells with VSVG trapped in the cell with and without FFAT and AAAT among the control and VAPs

2. Shear cells allowing vesicles to move away from cells

3. Add extra cytosol to keep necessary proteins

4. Low speed spin to remove large cells pellet from supernatant

5. Take pellet and do a 100000g spin

6. Spin supernatant

7. Check % of VSVG budding difference at 4C vs 32C

Results

With no peptide added to the cell in comparison to the control there is substantial decrease in % of VSVG budding among the VAPs

With the FFAT peptide added to the cell in comparison to the control there is relatively similar levels in % of VSVG budding among the VAPs

With the AAAT peptide added to the cell in comparison to the control there is a slight decrease in % of VSVG budding among the VAPs

The FFAT motif is the most helpful in improving movement of proteins through the ER into vesicles