Techniques: Proteins Flashcards
What is a Bradford assay?
Measurement of protein concentration
How does a Bradford Assay work?
Brilliant blue dye binds to protein and is measured to generate a number on a spectrophometer and then plotted on a standard curve.
In a Bradford Assay what colour is no protein?
Brown
In a Bradford Assay what colour is protein?
Blue
What do you need to do a Bradford Assay?
Blanks
Diluting Solution
A sample of known protein concentration
What is SDS-PAGE used for?
Visualising proteins
How to do a SDS-PAGE?
1) Denature Proteins
2) SDS binds giving a negative charge
3) Run through polychromide gel
4) Will travels based on mass
What is a Western Blot used for?
Visualising proteins
How to do a Western Blot?
1) SDS-PAGE
2) Blot proteins from SDS-PAGE onto membrane
3) Immunoblotting of protein
4) Antibody detection by adding 1st antibody
5) Add second antibody
6) Detect (wash often for clear results).
What do you need to be careful of when doing a Western Blot?
Load same volume
Controls
Primary antibody must be from a different species as the protein and second antibody (so three species).
What are different types of detection?
- Chemiluminescents
- Colormetric
- Fluorescent
- Radioactive
What is an ELISA?
Test of hormone
How to perform an ELISA?
Primary antibody binds to antigen
Secondary antibody binds to primary antibody
Enzyme binds second antibody turning blue
What colour is a negative ELISA?
Clear
What colour is a positive ELISA?
Blue
What is a direct immunohistochemistry and immunocytochemistry?
Antibody binds to antigen and has a fluorescent label
What is an indirect immunohistochemistry and immunocytochemistry?
Primary antibody binds to antigen.
Secondary antibody binds to antigen
Secondary has the fluorescent label
How do you perform an indirect or direct immunohistochemistry or immunocytochemistry?
1) Collect sample
2) Immunohistochemistry (same as ELISA)
3) Microscope and data analysis
What colour is a positive immunohistochemistry?
Brown - stained cells appear brown)
What do you need to an immunohistochemistry?
Positive control
No primary antibody
