Direct ELISA of Alpha-Amylase
Ellyn Daugherty
Welcome to my workshop:
SM Biotech Career Pathway
www.BiotechEd.com
ellyn@BiotechEd.com
www.sargentwelch.com/biotech
www.emcp.com/biotech

Fill in the card in the tri-fold brochure (if I don’t 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
In Biotech, companies find a product of interest
(amylase) and make sure to have assays to show it’s
presence, activity, & concentration
Amylase is an enzyme that
catalyzes starch digestion.
It is used commercially in
several ways including:
1) Remove starch in products
2) Produce sugar from starch
3) Processing of cellulostic
biofuel
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
Absorbance (a.u)
Ion Exchange Chromatography
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
-0.05 0
5
Fractions (#)
10
The San Mateo Biotechnology Career Pathway
ELISAs taught in Biotech 2 (Ch 6) and Biotech 4 (Ch 14)
The rAmylase Project
Model of rDNA/protein Business
< Chapters 1-5 Basic SLOP
< Lab 5f Amylase PAGE (revised)
< Lab 6e Amylase Producing Bacteria
< Lab 6c Amylase Activity Assay (revised)
< Lab 6d Amylase ELISA & Western Blot (new)
< Labs 7a/7b/7f/7g Amy Spectrophotometry
< Lab 8b Restriction Digestion of pAmylase
< Lab 4j/8b/8g DNA Gel Electrophoresis (revised)
< Lab 8c pAmy Transformation of E. coli
< Lab 9c/9d Amy Ion-Ex Chromatography
< Lab 8g/4h Genomic & plasmid DNA Isolation
< Lab 13i Amylase Gene PCR (new)
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
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.
Di
Lab 6d Amylase Elisa Kit Expected Results
<<<<< The Standards should look like this …
…and the unknowns should
look something like this >>>
Automated ELISA Plate Reader
Get more help…
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