Mammalian Flashcards
You are planning to prepare a protein extract from mammalian cells. You have the following lysis buffer. What is the function of each of the components in this buffer?
Non ionic detergent: Lyses the cells by breaking up the cell membrane, allowing us to extract the proteins
Sucrose: To keep the osmotic pressure at the same level, otherwise components of the cell might rupture when the cell membrane is broken
NaCl: Osmotic pressure, as above. No salt might cause cells to break once they take in too much water, same for other cell components
HEPES: Keeps the pH-level the same outside and inside of the cell, same reason as above, cells might break when they try to compensate for the different concentrations
Antigen bound to nitrocellulose membrane following SDS PAGE Small volume
Have the antigen that you use to purify bound to a piece of cellulose membrane. Run a cell extract SDS PAGE gel to separate by size. Then transfer the protein by western blotting to a nitrocellulose membrane and then cut out the tinny part that is the size. Use that as the source of antigen to purify the protein.
Protein A or G affinity purification
These are two proteins that are found in the cell wall of certain bacteria that bind to the conserved region of the antibody. That can provide affinity purification. You’re purifying something by the ability for it to bind to something else. You can bind it, wash away everything that doesn’t bind and release it. Works well on hybridermal cell lines. Only one type of cell and antibody. Monoclonal antibody
Original peptide antigen
Good for polyclonal antibodies. Need to be specific. Use your original peptide as a purifier. Same process…
What can you do if FRET does not work?
Reduce distance between fluorophores by directly couple fluorophores to the primary antibody.
Directly couple fluorophores to Fab fragments.
Two enzymes Pepsin and Papain. Cut antibodies. To get a small digest with papain.
Define the terms ‘monoclonal antibody’ and ‘polyclonal antibody
Give 3 advantage of a monoclonal compared to a polyclonal antibody
Polyclonal antibody- mixture of antibodies from an immune serum
Monoclonal Antibody- antibody secreted by a hybridoma clone
Single type of antibody
Easy to purify large amounts
Permanent reliable source of antibody
Single B cell line.
A single epitope targeted therefore more specific to a certain region of our POI.
You are at the start of a new project working on protein X. You want to purify the protein and also want to study its binding partners in mammalian cells. You have the choice of making a polyclonal or monoclonal antibody for your studies. Which would you choose and why?
I would choose to mate polyclonal antibodies. I would immunize a host organism e.g a rabbit with my protein-X. I would then be able to collect cells producing antibodies to all the antigen on my protein. In the purification step, this will mean that I hit multiple targets, epitopes, by using polyclonal. If i e.g want to target a specific part/fragment on my protein , but in this case. I believe polyclonal are the optimal choice. Polyclonal are also less time consuming to prepare.
If you are preparing a hybridoma cell line how do you select for the fused cells?
Need to explain that cells are present. Wait until the non fused b cells die. Mutation in the salvage pathway of the myeloma cells. Block synthesis explain that only fused cells can grow kill non fused myeloma cells.
Compare the benefits of using mammalian cells and E.coli to express your favorite protein.
Mammalian cells are good to choose if you want to express a complex and large protein. Mammalian cells can fold complex proteins and also do post translational modifications such as phosphorylation and acetylation. E.coli on the other hand lack the ability to do post translational modifications and complex folding. El.coli is a good option if you want a high yield of a protein which is not complex. Their cell cycle is short and they are cheap to use. Mammalian cells usually generate low yield of proteins, have a long cell cycle (24h) and are expensive to culture.
What is the difference between stable and non-stable transfections?
Transient:
Transient transfection cells express the forgin gene but do not integrate it into their genome. The new gene will not be replicated.
Stable:
Generating stably transfected cells begins with a transient transfection, followed by an infrequent but important and serendipitous process. In a small proportion of transfected cells, the foreign gene is integrated into the cells’ genome. The hallmark of stably transfected cells is that the foreign gene becomes part of the genome and is therefore replicated.
In what way are antibodies from alpacas and Llamas different from antibodies from mice?
Imagine you want to make an antibody to collect your protein of interest from cell extracts and then identify which proteins bind to. What are the advantages of using an antibody from an alpaca instead of a mouse?
Mice have “conventional” antibodies made from 2 heavy chains and 2 light chains. Alpacas have single chain antibodies made from 2 heavy chains. They are smaller and conventional antibodies but still have 2 binding sites. Since they only consist of 1 type of protein can be made easily in E.coli instead of B-cells (after isolation)
