Western blotting Flashcards
What is the purpose of detecting specific proteins?
- Seeing whether your protein is in your sample (not just at RNA level)
- If a process is occuring
- Can check size of protein and how much is there
- Can see where it is
How is western blotting similar to immunodetection?
- Specific antibody coupled to output
- Immobilistion
- Blocking
What is ELISA?
Using individual detection reactions contained in wells in order to quantify protein ammounts, very sensitive however protein size cannot be distinguised
What are the advantages of ELISA?
- Fast and cheap
- Can be automated easily
What is a dot blot?
Sample applied on membrane in a dot and then blotting procedure is performed
What are the advantages and disadvantages of a dot blot?
- Fast and allows bulk detection on the entire blot
- However is manual, not qualitative and only semi-quantitative
What are the advantages of a western blot?
- Semi-quantitative
- Can look at protein size allowing cross-reactivity to be distinguished
What is the protocol for a western blot?
- Prepare samples
- Run acrylamide gel to separate proteins
- Stain gel with coomasie stain to show bands
- Electroblot transfer of proteins from gel to membrane
- Stain membrane to mark positions of standards
- Block membrane
- Label with primary and secondary antibody
- Detect antigens
What is the purpose of SDS-page gel?
- Acts as a soap, has long lipophilic carbohydrate chain and polar sulfur group
- Unfolds protein and gives uniform negative charge allowing them to move across gel
- Movement completely based on molecular weight
- Without SDS charge interferes with this
What is the purpose of lamelli buffer?
- Contains SDS
- Contains blue stain to track progress
- Contains strong reducing agent to make sure all proteins are monomeric and completely unfolded
What is a coomasie stain?
- Dye that binds non-specifically to proteins, useful to estimate relative ammounts
- Dye not bound to protein diffuses out after destain steps
