Lab. Meeting (18th July)
In Vitro studies to define the role of PERK in insulin synthesis
* Using the 832/13 cell line
Experimental Design
Equal numbers of 832/13 cells were plated on dishes These were starved for the sulphur containing amino acids
methionine and cysteine for half an hour
Equal amounts of S35 was added to each dish
The cell lysates were harvested at different time points
(This would be indicative of radiolabelling in the intracellular compartment)
Determination of time point of maximum S35 incorporation
S35 Incorporation: Total Protein
0
5
10
15
20
25
30
35
Time
Perc
enat
ge
Inco
rpor
atio
n
Series1
Studying protein synthesis (intracellular) using S35 labeling
832/13 cells
832/13 cells with lacZ
832/13 cells with ▲C
INCUBATED for 36 hours, no significant cell death
at the time of harvesting
= Untreated controls
= (adenovirus vector without the dominant negative construct)
=(adenovirus vector with the dominant negative construct)
S35 incorporation following 30 mins of pulsing
0
5
1015
20
25
3035
40
45
% incrprn.
s35 incrprn final & revised
Untreated lacZ ▲C
Labeling as a fraction of total Protein content in samples
0200400600800
10001200140016001800
ug/800ul cell lysate
labelled fraction totalprtn.n
total protein
Untreated LacZ ▲C
Autoradiograph for a western of the same
Next step
Immunoprecipitation for insulin Comparing Insulin levels from different cell
lysate samples