Lecture 9 Flashcards
How does Polyacrylamide Gel Electrophoresis work?
- used for analytical separation of proteins, when one doesn’t need a large amount of protein
- positive molecules migrate to cathode and negative molecules migrate to anode
– Molecules will migrate through along an electrical field though a gel matrix according to their size, shape, and charge
– Large molecules are impeded, while smaller ones slip through the matrix more easily
Whats the purpose of the buffer solution in the gel electrophoresis?
– generates the current between the anode and cathode
How does SDS-Page: Denaturing Gel work?
– SDS denatures proteins, causing multimeric proteins to dissociate into their individual subunits
– SDS coats all the polypeptide chains with a negative charge, therefore forcing the polypeptide chains into extended conformation with similar charge: mass ratios (getting rid of charge)
– SDS eliminates the effect of differences in shape, and so chain length (mass) is the sole determinant of the migration rate in SDS-gel electrophoresis
– this method aids with eliminating the two obstacles of shape and charge when it comes to trying to isolate proteins
What are the different reagents that denature protein and what do they do specifically? Which of these are reducers?
- Urea
- Guanidinium chloride
- Beta mercaptoethanol
- Sodium dodecyl sulfate (SDS)
- reduces disulfide bonds in particular
- these reagents eliminate those bonds that are often important for folding up 3-D structures in proteins
Reducer: Beta mercatoethanol
How does SDS-PAGE work?
– the sds is poured into solution that will unfold the proteins and denature them, making them pretty linear and turning multimeric proteins into their monomer forms.
– Because SDS is really negatively charged, each protein being studied will be coated in negative charge so no other charge will be present.
– All proteins will then move towards the positive anode.
– Because of this, all proteins will have a similar mass-charge ratio, eliminating the effect of shape and charge of proteins in separation.
–Separation will only depend on size (also known as molar mass. smaller molecules will move more quickly in gel and be at the positive (anode) end and larger ones won’t be as far
T or F, SDS-PAGE isolates proteins through eliminating charge, shape and size.
False, SDS-PAGE isolates proteins through eliminating charge and shape
T or F, in SDS-PAGE, proteins are coated with a detergent, SDS, which denature (unfolds) then and coats with a negative charge. Thus they only separate only according to smaller proteins move more quickly towards the anode than large ones.
True
T or F, non reducing conditions means that disulfide bonds remain unbroken (disulfide bonds)
True
T or F, without a marker lane for SDS-PAGE, one has no idea the size of one’s proteins
True; marker lane tells us approximate size of proteins in sample lane
How does SDS page in reducing conditions work?
It works by adding beta mercaptoethanol that reduces disulfide bonds; remember out of all the reagents that denature proteins this was the one that reduces and within this context reduction pertains toward disulfide bonds
What do darker dots in SDS page show?
The relative amount of that protein in the solution –> darker it is the more protein there is there
If you are given an unknown protein and asked to identify it, how could you use SDS page?
– you run an sds page and if that protein falls in a range, you would see which proteins on the reference column fall in that range.
– this will probably give an indication that the unknown protein is the same as the distance moved by that protein in the reference page.
Describe a western blot.
– type of SDS page that focuses on separating proteins based on the use of antibodies.
- Substrates Will bind at the antigen side on the terminal heavy light chain.
- Binding of protein to the antibody and seeing reaction indicates correct match.
- It mainly separates by size and identity because of specific binding to certain known antibody
Why is protein transferred to a polymer sheet? And Why is a second antibody added during Western Blotting?
– because antibodies won’t be ab;e to bind to proteins w/in gel matrix
– because produces a color light when presented with a substrate
How does isoelectric Focusing and SDS page work aka 2D-Gel electrophoresis?
– it separates proteins based on their charge.
– proteins with higher isoelectric points will tend to move further down the column towards the high pH scale (moves from low pH to high pH).
– Once separation by charge occurs, it is ran through an SDS and further separated by size
– Essentially, separation by charge then by size (molar mass)
How is 2-d Electrophoresis ran spatially?
the isoelectric focusing is ran horizontally and the SDS page vertically
What is a good example of use of 2-d electrophoresis
- It can be used whether deficiencies or excess in a normal vs abnormal cells exist in terms of protein expression.
- If something is under or over expressed in the abnormal cell, it can be used for the target of drugs.
True or false
enzymes increase the rate of biochemical reactions
True
