BMD RES 5HA Lecture Notes - Lecture 8: Primary And Secondary Antibodies, Chemiluminescence, Membrane Protein

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7 Feb 2017
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Immunoblot/western blot
Darker band: more of the protein
Use antibodies (immuno) as part of the technique
The cells have so much of actin and myosin in muscle
cells that the proportion of DGC is very small
The antibody will find the DGC, but the camera
may not be able to detect the signal
Step 1: Enrich the proteins in interest and fish it out
The DGC has sugars unlike most proteins
Dr. Gibbs added a bead coated with something that
likes to stick to sugars (SWGA—wheat germ
agglutinin—a type of lectin—a protein that binds
sugars)
Increases the proportion of DGC
Only because DGC is a membrane protein, and
those are in small proportions in muscle cells
To identify a specific type of protein, we will use this
technique
First, place a "membrane" on the surface of the gel and
apply an electrical current to move the proteins onto the
membrane
Second, remove the gel and discard. The proteins should
now be firmly attached to the membrane
The ladders are usually pre-stained so that they will
show colored bands at particular sizes right on the
membrane
Third, we will now add a primary antibody against our
proteins of interest
Fourth, add the secondary antibodies that recognize the
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