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

Lab. Meeting (1 st August) PowerPoint PPT Presentation


  • 39 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 1 st august

Lab. Meeting(1st August)

  • In Vitro studies to define the role of PERK in insulin synthesis

    * Using the 832/13 cell line


Experimental design

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

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 incorporation1

Determination of time point of maximum S35 incorporation


Studying protein synthesis intracellular compartment 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)

Studying protein synthesis (intracellular compartment) using S35 labeling


S35 incorporation following 30 mins of pulsing

S35 incorporation following 30 mins of pulsing

Untreated lacZ ▲C


Labeling as a fraction of total protein content in samples

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

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


Immunoprecipitation for insulin

Immunoprecipitation for insulin

Untreated LacZ ▲C

Scintillation counter readings


Immunoprecipitation for insulin1

Immunoprecipitation for insulin

Untreated LacZ ▲C


Immunoprecipitation for insulin non reducing gel without urea

Immunoprecipitation for insulinNon Reducing gel (without Urea)

Untreated LacZ ▲C

Non Specific Aggregates?


Immunoprecipitation for insulin reducing gel with urea

Immunoprecipitation for insulin Reducing gel (with Urea)


Immunoprecipitation for insulin2

Immunoprecipitation for insulin

Untreated LacZ ▲C

Suggests higher insulin synthesis in delta C treated cells


Insulin content

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

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