Development of western blots for actin without the use of radioactivity
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Development of Western Blots for Actin without the use of radioactivity. Geoff Theobald STEP Summer Internship Program June 2003. Objective. To create a Western Blot without the use of radioactivity. Significance. Safety & disposal

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Development of western blots for actin without the use of radioactivity

Development of Western Blots for Actin without the use of radioactivity

Geoff Theobald

STEP Summer Internship Program

June 2003


Objective
Objective radioactivity

To create a Western Blot without the use of radioactivity.


Significance
Significance radioactivity

  • Safety & disposal

  • Use in college lab: students learn about gene expression and molecular biology


Background information
Background Information radioactivity

Actin:

  • Is the protein we detected using a Western Blot.

  • Has a molecular weight of 42,000-43,000 daltons.

  • Works with myosin (another protein) in muscle contraction.

  • Is also involved in the structure in most cells.


Methods
Methods radioactivity

  • Polyacrylamide gel electrophoresis

  • Western Blot

  • Probing with an antibody to actin

  • Detection by fluorescence


Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis radioactivity

  • Separates proteins by size

  • Protein standards are used to determine the size of Actin


Western blot

Pic. Of membrane radioactivity

Western Blot

  • A western is when you transfer protein out of polyacrylamide gel and into a membrane.

  • A Western Blotis the result of the transfer.

Gel


Probing with an antibody to actin

C-term radioactivity

N-term

Actin Protein

Probing with an antibody to actin

Probe blot with antibody


Detection by fluorescence

DDAO radioactivity

DDAO phosphate

Alkaline Phosphatase

Key

Secondary Antibody for Rabbit

= Actin

= Primary Antibody

= Secondary Antibody

Primary Rabbit Antibody for Actin

= DDAO

= DDAO phosphate

= Alkaline phosphatase

Actin

Western Blot

Detection by fluorescence


Results detection with uv light

Pic. of Blot exposed to UV light radioactivity

Results: detection with UV light

  • There was no clear signal.


Fluorescent dye can be visualized with uv or white light
Fluorescent dye can be visualized with UV or white light radioactivity

Advantages

  • UV: we can distinguish red light

  • White light: stronger excitation


Absorption emission spectrum of ddao
Absorption/emission spectrum of DDAO radioactivity

  • Use white light with a blue filter


Results detection with white light

Pic. Of blot exposed to white light radioactivity

Results: detection with white light

  • There was no clear signal.


Chemiluminescence assay

Pic of dot blot with all actin antibody radioactivity

Pic of membrane with all actin antibody

Chemiluminescence assay

  • Worked well. Continued experiments with this assay.


Conclusions
Conclusions radioactivity

  • Detection of fluorescence with UV light did not work.

  • Detection of fluorescence with white light and a blue filter did not work.

  • Since fluorescence assay did not work well, but chemiluminescence worked, we will concentrate on that assay.


Acknowledgements
Acknowledgements radioactivity

  • Dr. Guzman, Biology Dept.

  • Dr. Metz, Biology Dept.

  • Mrs. Bloom, STEP program

    I would like to thank each and everyone of you for all of your advice, support, and fun I had these past two weeks.


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