experiment 7: western blot analysis of rGFP fractions Flashcards
what is the main difference between staining and immunological detection?
- staining: non-specific, will bind to all proteins using a dye such as Coomassie Blue
- immunological detection: is specific (binds to one protein) and uses antibodies to bind to an epitope tag (such as chromophore) on an antigen (ex: western blot)
what are the types of membranes discussed in class? why is one chosen over the other?
nitrocellulose: used for general western blotting, has a low tensile strength (breaks easily) and a low protein binding capacity
PVDF: expensive, high chemical resistance, tensile strngth, high protein binding and tenetive capacity, can be handled repeatedly (re-probing) and is good for sequencing
nylon: expensive, strong synthetic polyamide sheet, can be re-probed
what is the purpose of incubating the membrane with BSA, gelatin, or a mixture of dry milk?
it helps block any non-specific binding to the protein. it also saturates all of the sites on the nitrocellulose membrane not already bound with protein. if there are sites not blocked, the primary and secondary antibodies would bind non-specifically throughout the membrane making separation of protein of interest difficult
what is the difference between a primary and secondary antibody?
primary: raised against antigen, binds to protein of interest to allow for its detection. in the lab we used mouse IgG anti-Xpress epitope monoclonal antibody raised against the “Xpress” epitope
secondary: binds to primary antibody, amplifies the signal provided by primary. in lab we used sheep anti-mouse IgG polyclonal anti-serum conjugated horse radish peroxidase
what is the purpose of the Tween 20 used in the wash steps?
tween 20 is a non-ionic and non-denaturing detergent that helps disrupt non-specific hydrophobic interactions b/w the primary antibody and other proteins on the membrane that formed during incubation. it helps to remove unbound antibodies from the membrane while also reducing the unspecific background of the membrane.
what is the advantage of using a colorimetric enzyme conjugated to a secondary antibody as opposed to being conjugated to the primary antibody?
multiple secondary antibodies can bind a single primary antibody, the protein of interest can only bind one primary antibody. so, having the colorimetric enzyme conjugated to a secondary antibody provides signal amplification as opposed to being conjugated to the primary antibody
what are the advantages and disadvantages of using a chemiluminescent reaction versus a colored precipitate reaction?
- chemiluminescent: monitors light emission, provides easy way to measure quickly, intensity can be measured on film (HRP and AP), if using PVDF membrane. need a dark room and/or developing solutions, expensive, more sensitive than colored precipitate (HRP), can be overexposed as many times as needed
- colored precipitate: precipitate formation, TMB with HRP precipitates purple, gives easy way to visualize clearly and see relative effects. overexposure for colored precipitate can ruin the proteins on the membrane
how are antibodies named?
primary antibody that binds to the antigen: Rabbit anti(α)-GFP polyclonal anti-serum (it must be anti GFP in order to bind to the GFP Antigen of interest. Whatever follows the “anti” is what it is recognizing). “Rabbit” at the beginning is the animal that the antibody was raised in by injecting it with GFP.
secondary antibody that binds to the primary antibody: Goat anti(α)-rabbit IgG polyclonal anti-serum conjugated AP. The secondary antibody must be anti-rabbit in order to recognize the rabbit antibody. The host that the antibody was raised in is a goat. Conjugated AP is a detection method.
- Antigen: His6/Xpress Epitope/GFP (our lab)
- Primary Antibody: Mouse anti(α)-Xpress MAb Monoclonal Antibody
- Secondary Antibody: Sheep anti(α)-Mouse IgG Polyclonal anti-serum Conjugated HRP
what is the difference between monoclonal and polyclonal antibodies?
polyclonal: contains many antibodies that target different epitopes on the target protein
monoclonal: single type of antibody that targets a single epitope antibody, expensive
given several antibodies, determine which ones would be used in which order to develop a western blot
antibody that works against the protein of interest **(anti-rGFP).
Second antibody works against the primary antibody and contains a bound enzyme to allow for enzymatic detection (anti-mouse).
define epitope
short sequences on an antigen 8-10 amino acids long, sequence is physically recognized by an antibody
define antigen
a foreign particle that elicits an immune response
define antibody
a protein generated by the immune system in a reaction to a foreign substance
define antiserum
blood serum containing polyclonal or monoclonal antibodies that target the protein of interest
define serum
a fluid and solute component of blood (control, no protein of interest)
