# Protein Analysis To properly study a protein, it must be available at a high concentration from a source material, meaning that [[Protein Purification]] is often the first step taken **Forward genetics** analyzes *existing proteins* to identify an associated gene **Reverse genetics** analyzes *phenotypes* after starting from an approximate location to identify an associated gene | Technique | Type | Description | | ------------------------------- | ---------------------------- | ------------------------------------------------------------------------------------ | | EMSA | | | | Protease Digestion | | | | Far-Western Blot | Protein-Protein Interactions | Uses a native gel and radiolabeled bait protein probe, detected with autoradiography | | Footprinting | | | | Yeast Two-Hybrid | | | | Calorimetry | Protein-Protein Interactions | Measures minute changes in heat that indicate binding or conformational change | | ChIP | | | | [[Mass Spectrometry]] | Protein | | | Surface Plasmon Resonance (SPR) | | | | Southwestern-blot | | | | Edman Degradation | | | ![[Pasted image 20230310231914.png|300]] ## Analyzing Protein Structure - [[X-Ray Crystallography]] ### Sequence Analysis It’s important to remember that once the genetic material has been [[Translation|translated]] into a polypeptide chain, the process is not reversible (due to the # of amino acids not aligning to the # of unique codons) ![[Pasted image 20231016142442.png|400]] Cyanogen Bromide: Cleaves at methionine Ser - Tyr - Ser - Met - Glu - His - Phe - Arg - Trp - Gly - Lys - Ala - Val #### Edman Degradation (N-Terminal Sequencing) Edman degredation - Developed by Frederick Sanger in 1950s to sequence - [[Bioinformatics]] ## Analyzing Protein Interactions - [ ] Analyzing Protein Interactions - [ ] Define techniques used to identify protein-protein interactions - [ ] Pull-down assay - [ ] Yeast two-hybrid - [ ] Far-western blot - [ ] Microarray - [ ] Tandem affinity - [ ] Calorimetry - [ ] Surface plasmon resonance - [ ] [[Analyzing Protein-DNA Interactions]] - [ ] Define techniques used to identify protein-DNA interactions (southwestern blot, EMSA, DNA footprinting, chromatin immunoprecipitation). - [ ] Given an EMSA, determine which proteins interact with the DNA of interest. ## DNA-Protein Interactions Studying DNA From Proteins** In the early days of molecular biology, accurate back-translation from a protein sequence to DNA was nearly impossible. In modern times, many techniques exist such as Edman degradation that allow us to sequence the If an antibody had a high enough affinity, it could potentially bind to the protein’s polypeptide chain immediately after it leaves the ribosome (still during the process of translation). This would form a complex with the polypeptide, the ribosome, and the mRNA fragment being read, which could then be cloned and sequenced to identify the DNA origin of the protein. - *Sanger Sequencing:* 4 tubes each containing their own type of ddNTP which terminates the synthesis process once added. This results in a series of bands being generated, indicating - *Maxam-Gilbert Sequencing:* aaa --- - *Restriction Fragment Length Polymorphisms (RFLP):* Relies on the site-specific cleavage of restriction enzymes to target locations associated with single nucleotide polymorphisms. The presence or absence of specific fragments when run on a gel reveals the genotype of the individual at the SNP site. - Can be used to conduct linkage mapping when combining different samples who contain different SNPs - Southern Blot: Combines the techniques of restriction enzyme digestion, gel electrophoresis, and hybridization using a probe ![[Pasted image 20240427025722.png|400]]