I need to make an antibody for the extracellular portion of the Notch receptor. I have made S35 labelled Delta and my boss has given me an antibody that recognizes Delta and is conjugated to a chromophore that gives off light. Place the following statements in order beginning with labeling. HINT: Labeling, protein enrichment, immune response, making monoclonal cell lines, antigen screening of monoclonal cell lines.
A) Hybridoma cells are selected by plating them in HAT buffer.
B) Using direct ELISA you coat individual tissue culture wells/dishes with a single monoclonal antibody.
C) I detect my protein fraction using a geiger counter.
D) You add a solution containing radiolabeled Notch extracellular domain to the wells.
E) You only keep monoclonal hybridoma cell lines that give light. The other cell lines you throw away. You have your antibody!
F) I centrifuge my homogenate in a sugar or salt gradient.
G) I inject a mouse with my protein, giving booster shoots every week.
H) Heterokaryon cells are generated by the fusion of plasma cells and a myeloma cell line.
I) You wash away excess Delta antibody.
J) You add medium containing the Delta antibody that is covalently bound to a chromophore to the individual wells of the ELIZA.
K) To determine if the monoclonal antibody from any given monoclonal cell line recognizes the Notch extracellular domain, you look for light.
L) I use dialysis to remove excess salt and or sugar and to place the protein into a solution that mimics that of the cytoplasm.
M) The mouse is euthanized to harvest the plasma cells in the spleen.
N) Vast quantities of the exported form of the triggered antibody from plasma cells are released.
O) On some B cells, individual antibody receptor forms are bound by the Notch antigen for and activate B cells.
P) I fractionate the proteins.
Q) Individual hybridoma cells are cultured in separate tissue culture wells/dishes to create monoclonal cell lines. These lines export the unique antibody they make into the culture medium.
R) I take culture cells with the Notch receptor expressed on the cell surface and mix them with radio-labelled Delta.
S) B cells divide to give rise to plasma cells and memory cells.
T) You wash away excess Notch extracellular domain from the coated ELISA wells
