GFP is a protein that glows bright green in the presence of UV light. GFP can be translationally fused to a variety of proteins, and because the protein of interest (in this case BRI1) has GFP bound to it, it can also be detected by irradiation with UV light. Chimeric genes, consisting of GFP linked to genes of protein of interest, can be created. GFP It is introduced into organisms and maintained in their genomes. However, the BRI1-GFP protein needs to be taken out of solution for biochemical analysis. This is done by immunoprecipitation:
Anti-GFP antibody was added to the mixture, which bound to the BRI1-GFP fusion protein. Now we have BRI1-GFP-antiGFP. In order to precipitate this out of solution, Protein A was added. Protein A is a protein that binds to antibodies. Protein A bound to the anti-GFP part of the BRI1-GFP-antiGFP complex, pulling the complex out of solution. The proteins were separated using gel electrophoresis, and then stained with Coomassie stain. The Coomassie stain is a method that adds class-specific dye (in this case, protein-specific) to a substance to qualify or quantify the presence of a compound.

The researchers made BRI1-GFP proteins in Arabidopsis, and then pulled out the protein by immunoprecipitation. They then analyzed the profiles of the immunoprecipitated proteins, and found that BRI1-GFP was the main precipitated protein.

**tritium=isotope of Hydrogen with 3 neutrons.
GST has a high binding affinity for glutathione, and when a fusion protein is exposed to a glutathione resin, the complex will be drawn out of solution because the GST part binds to the resin. 

GST-BRI1 fusion proteins were made, and then the solution was incubated with tritium- labeled brassinolide, which hypothetically would bind to BRI1. GST has a high binding affinity for glutathione, and when the fusion protein was exposed to a glutathione resin, the complex was drawn out of solution because the GST part bound to it. The GST-BRI1 complex was then analyzed to see if there was tritium labeled BL bound to BRI1.

Background info on constructs

Eukaryotic DNA can be expressed in prokaryotic bacteria. The DNA of interest can be cloned in an expression vector (put into a bacteria plasmid). How constructs are made:
There is a restriction site on the plasmid where the DNA binds. Upstream to it, there is a promoter site, where RNA polymerase initiates transcription, and a ribosome-binding site, where translation occurs. Promoters drive the synthesis of foreign proteins in the plasmid. The tac promoter was used in this experiment, which is a hybrid of the lac and trp promoters. The operator was the lac operon, which controls expression of the cloned inserts. The repressor was a variant of the wild type lac repressor, and it prevented premature gene expression. The tac promoter allowed for chemically inducible, high levels of expression of the BRI protein. Expression vectors have DNA inserts cloned into the coding sequence of a protein-coding gene in the host cell. The recombinant gene sequence consists of the coding region of the host cell and the cloning site of the new DNA. Translation occurs, and the protein synthesis mechanism of the host cell synthesizes constructs from the host cell DNA and the foreign DNA inserted into the plasmid. Protein expression was driven by the host RNA polymerase using the tac promoter. Five DNA sequences were expressed with GST and tested for BR binding:

1. ID
2. LRR21-ID-LRR22
3. LRR21-ID
4. ID-LRR22
5. LRR22

BRI1 complementary DNA was cloned with GST into the BAMHI site of pGEX-5X. This is an expression vector of E.coli, and researchers put BRI1 DNA from a plasmid into an E.coli cell, and the replication mechanisms of the E.coli cell produced BRI1 protein. BRI1-GFP fusion proteins were expressed in E.coli expression vector.

Crosslinking Reaction of a Simple Aryl Azide: http://probes.invitrogen.com/handbook/figures/0222.html
Photoreactive crosslinking probes are used to determine the relationship between two reactive groups, in this case one on a ligand and the second on its receptor. The crosslinkers are chemically reacted with one molecule, and then the molecule is reacted with a second molecule using UV light. The chemically modified molecule was BPCS, and the second molecule was the BRI1 receptor. This allows joining of the two molecules by a covalent bond.

Western blotting determines the molecular weight of a protein, and the relative amount present using an antibody. First, samples are prepared from tissue/cells that are homogenized in a buffer that protects the protein of interest from being degraded. The proteins are separated by SDS-PAGE. This uses gel electrophoresis, which separates proteins by size. SDS (sodium dodecyl sulfate) dissolves cell membranes and denatures proteins into their linear structures. It also coats the protein with negative charges because the protein will be transferred to a polarized gel. PAGE is a polyacrylamide gel that the protein is transferred to after it is treated with SDS. It binds any sticky places on the protein. A primary antibody is added to solution that binds the protein of interest. A blocking buffer must be added to prevent the primary membrane from interacting with the gel. The buffer used is typically a solution of protein (dry non-fat milk or Tween). A secondary antibody-enzyme conjugate is added that will bind to the primary antibody, and this will show where the primary antibody bound, identifying the protein of interest. The proteins are then run through the PAGE gel where they are separated by size. The proteins are then transferred to a membrane (nitrocellulose or PVDF). The membrane is placed face-to-face with the gel, and a current is applied. The charged proteins move from the gel to the membrane. The purpose of transferring to a membrane is to expose the proteins to a thin surface for simpler detection. The membrane has the bands of proteins on it, and the protein of interest is identified.