Ellyn Daugherty

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Direct ELISA of Alpha-Amylase

• Ellyn Daugherty
• SM Biotech Career Pathway
• www.BiotechEd.com
• ellyn_at_BiotechEd.com www.sargentwelch.com/biotech
• www.emcp.com/biotech

• Welcome to my workshop
• Fill in the card in the tri-fold brochure (if I
  dont already have one from you) and turn it in
  to me a copy of my curriculum.
• Please read through these slides (on the handout
  in your pee-chee folder).
• When done, please read through the lab packet (in
  the pee-chee folder).
• -) Ellyn

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In Biotech, companies find a product of interest
(amylase) and make sure to have assays to show
its presence, activity, concentration

• Amylase is an enzyme that catalyzes starch
  digestion.
• It is used commercially in several ways
  including
• Remove starch in products
• Produce sugar from starch
• Processing of cellulostic biofuel

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The rAmylase Project is the focus of the 2nd
semester of the SMBCP

• Learn how to assay for a protein of commercial
  interest (amylase)
• Transform cells to produce that protein (using
  pAmylase and C2523 cells)
• Scale-up cells to volumes where amylase can be
  purified and assayed

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The San Mateo Biotechnology Career Pathway
ELISAs taught in Biotech 2 (Ch 6) and Biotech 4
(Ch 14)
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The rAmylase Project Model of rDNA/protein
Business
lt Chapters 1-5 Basic SLOP lt Lab 5f Amylase PAGE
(revised) lt Lab 6e Amylase Producing Bacteria lt
Lab 6c Amylase Activity Assay (revised) lt Lab 6d
Amylase ELISA Western Blot (new) lt Labs
7a/7b/7f/7g Amy Spectrophotometry lt Lab 8b
Restriction Digestion of pAmylase lt Lab 4j/8b/8g
DNA Gel Electrophoresis (revised) lt Lab 8c pAmy
Transformation of E. coli lt Lab 9c/9d Amy Ion-Ex
Chromatography lt Lab 8g/4h Genomic plasmid DNA
Isolation lt Lab 13i Amylase Gene PCR (new)
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Introduction to ELISA
ELISA short for enzyme-linked immunosorbant
assay
An ELISA uses antibodies with conjugated enzymes
to specifically identify and measure the amount
(concentration) of a protein in a solution
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• ELISA
• A specific antibody (Ab) recognizes a specific
  protein (Ag).
• An enzyme bound to the Ab causes a color change
  in a substrate (so you can see the Ab is
  there).
• The more color in a well, the more Ab-enz complex
  is bound to target protein higher target
  protein concentration.

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Lab 6d Amylase Elisa Kit Expected Results
ltltltltlt The Standards should look like this
and the unknowns should look something like this
gtgtgt
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Automated ELISA Plate Reader
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