By Emily Johnson

1
Development of a Western Blot For Actin Using
Chemiluminescence

• By Emily Johnson
• Summer STEP Program, 2003

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Objective Develop Non-radioactive Western

• Specific Aims
• Show we can use the actin antibody to
  specifically recognize actin
• See if the antibody will recognize the protein
  produced in a test tube
• Show that antibodies to certain parts of the
  actin protein will also specifically recognize
  actin

3
Significance

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

4
Background Information

• Gene Expression

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Antibodies That Detect Actin
C-term
N-term
Key
antibody to entire actin
antibody to N-terminus of actin
antibody to C-terminus of actin
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Methods
www.sdsc.edu/Publications/ScienceAlive/
A Picture of Protein
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Polyacrylamide Gel Electrophoresis

• Process of separating the protein by size
• Protein standards are used to determine the size
  of actin

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Western Blot

• Transferring from a gel to a membrane

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Probing

• Antibody sticks specifically to actin

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Detection By Chemiluminescence

• Flow Diagram

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Principles of Chemiluminescence
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Results
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Does the Actin Antibody Specifically Recognize
Actin?

• Binds to actin, but not another protein called
  BSA
• Uneven spreading of ECL reagent

• Binds to a band at 42-43kd
• Binds to the band seen with pure actin
• A little binding to markers
• Non-specific?
• Something similar?

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Do Antibodies, to Certain Parts of the Actin,
Specifically Recognize Actin?

• All 3 antibodies have low binding to BSA
• Unsure why,but , in general, some dots dont
  change with concentration
• Technical?
• Blots show strong signal of actin protein

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Does the Antibody Recognize the Protein Produced
in a Test Tube?

• Several bands bind each of the three antibodies
• Binding IV, some of expected size, but need
  control reaction without gene
• IV ? 3 main bands
• Interesting is smaller
• Band not bound by
• C-term antibody

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Conclusions

• The actin antibody to the entire actin
  specifically recognizes actin.
• The antibody seems to recognize actin produced in
  the test tube. More experiments are necessary.
• The antibodies to certain parts of the actin do
  specifically recognize actin.

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Acknowledgements

• Dr. Guzman-Biology Department
• Mrs. Bloom-STEP Coordinator
• Dr. Metz-Biology Department
• Campbell University
• All the people who created the STEP program
• Thank you for all your help with our experiments
  and presentations. Without you none of this would
  be possible for us.

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The Western!!!