Lab meeting 1 st august
Sponsored Links
This presentation is the property of its rightful owner.
1 / 15

Lab. Meeting (1 st August) PowerPoint PPT Presentation


  • 48 Views
  • Uploaded on
  • Presentation posted in: General

Lab. Meeting (1 st August). 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

Download Presentation

Lab. Meeting (1 st August)

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Lab. Meeting(1st August)

  • 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

  • Methionine and cysteine labeled with S35 was added to this medium (should be incorporated in any new proteins synthesized)

  • The cell lysates were harvested at different time points

    (This would be indicative of radiolabeling of proteins in the intracellular compartment)


Determination of time point of maximum S35 incorporation

  • Comparison of beta emission generated signal from

  • Total cell lysates

  • (TCA precipitated) Total protein (from the cell lysates)

    : Gives data regarding percentage incorporation


Determination of time point of maximum S35 incorporation


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)

Studying protein synthesis (intracellular compartment) using S35 labeling


S35 incorporation following 30 mins of pulsing

Untreated lacZ ▲C


Labeling as a fraction of total Protein content in samples

Untreated LacZ ▲C


Autoradiograph for a western of total protein (TCA precipitated samples) to check for signal


Immunoprecipitation for insulin

Untreated LacZ ▲C

Scintillation counter readings


Immunoprecipitation for insulin

Untreated LacZ ▲C


Immunoprecipitation for insulinNon Reducing gel (without Urea)

Untreated LacZ ▲C

Non Specific Aggregates?


Immunoprecipitation for insulin Reducing gel (with Urea)


Immunoprecipitation for insulin

Untreated LacZ ▲C

Suggests higher insulin synthesis in delta C treated cells


Insulin Content

  • Daorongs data suggests that

    - 1. Insulin content in delta C treated cells is lower

    2. Delta C treated cells secrete lower amounts of insulin when stimulated with secretagogues

  • My data suggests that

    1. Global protein synthesis is reduced

    2. Insulin synthesis is increased


Next Step

  • More replicates

  • Check insulin content, too for untreated and vector treated controls for insulin content under similar conditions

  • IP another abundant protein


  • Login