D103 Review study Guide 2024

Principles of immunofluorescence staining we want to identify 2 dif proteins, how do we do it? *Make sure that ........ (2 essential parts to note) - First we must generate primary antibodies that recognize/bind our target protein from 2 DIFFERENT species animals. then, use secondary antibody (tagged with color) from a 3rd animal that recognizes/binds the primary but is a dif color than the other secondary antibody *primary antibodies must be from different species *secondary antibodies must be a dif. color than each other, must be dif animal than primary animal, but can be from same animal as other secondary so basically, at least 3 dif animals required, refer to slide for better understanding rabbit ;;;;;;;;;;;;;;;;;;;;;;;;;;;;mouse goat ;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;; goat how and what is Tagging used for? - through protein fusion of a fluorescent protein, we can "tag" a protein of interest/target, making it fluorescent, so that we can see/monitor behavior GFP - green fluorescent protein, used for tagging, when shine blue light on it, reflects & fluoresces green light back Many types of tags and colors, name 2 examples please - GFP: green fluorescent protein RFP: red fluorescent protein BFP: blue fluorescent protein, etc Immunofluorescense staining can be compared to a ___________ while Fluorescent tagging can be compared to a ____________. - -snapshot -movie Keep in mind that... - a movie can be made of different snapshots so tagging can also be used for immunofluorescent staining another tagging technique used to observe protein dynamics is FRAP. what's that? - -Fluorescent Recovery After Photobleaching -where they take a protein expressing GFP, and shoot a laser at a specific part, to determine how fast the GFP signal/protein heals at that part, which shows how dynamic the protein is! (similar to the scratch 'n' heal method) Understand FRAP chart - photobleaching disrupts GFP protein signal, as time passes measures how fast the protein signal can be recovered to tell whether a protein is highly or low dynamic fast recovery= high dynamics slow recovery=low dynamics btw dynamic= unstable (i think) *the lower the bar the lower dynamic, *the vertical line down represents 0 dynamics bc thats the photobleaching happening Western blot assay - technique that shows protein molecular weight shows when the size changes either increase or decrease as compared to each row, must analyze to find answer refer to i-clicker question and practice Cell sizes: bacteria , yeast , oocyte , epithelial largest? smallest? in between? - largest: Oocyte smallest: bacteria in between: yeast, epithelial, major difference between prokaryotic and eukaryotic cells? - eukaryotic contain organelles, prokaryotic no organelles to visualize/see small things we use a ____________. Some microscopes have limitations due to _____________ and source ___________ capability.