QBM CH 9
Bovine serum albumin weight
67kDa
Where is the protein of interest on an SDS PAGE?
Consistant, last protein eluted
Can proteins bind differently to SDS?
It is rare but some do bind SDS differently
Sample loading buffer is also known as
Laemmli Buffer
Advantages of TCE?
Quick detection (2 minutes) and microgram sensitivity bands are not fixed to gel so they can be used for other techniques
What is added to the stacking gel?
Tris-HCl pH 6.8
What is added to the resolving gel?
Tris-HCl pH 8.3
What is a gradient gel?
a gel that can be used in which the acrylamide concentration increases towards teh bottom of the gell
What is 2,2,2-trichloroethanol?
a molecle that will react with tryptophan under UV light
Advantages and disadvantages of silver staining
ad: high sensitivity dis: time consuming, difficult, expensive and has toxic biproducts
How does SDS denature proteins?
by breakng weak interactions and causing the protein to unfold and linearize due to repulsion
Advantages and disadvantages of B-ME
cheaper but more volatile and less potent
Why is the polypeptide chains more negative than normal?
each SDS molecules has 2 negative charges at pH values
How sensitive is SDS PAGE?
enough to resolve between 2 proteins that are approximately 1% apart in mass
Where are the most contaminants found
in the first few lanes
What is SDS PAGE used to determine?
weight, identity, purity and quantity
Can pore size be adjusted any other way?
yes, by adjusting the amount of acrylamide/BIS crosslinking
If proteins of a low weight are being used, the gel should have a ___ % of acrylamide
higher
Size of lysozyme
14 dKa
Which proteins migrate first, large or small?
Small! Large pores can not pass through
What does the pH difference generate?
an ion gradient via glycine and Cl- which carry the current and assist in the stacking process
Ammonium persulfate (APS)
necessary to catalyze the reaction between acrylamdie and BIS
The migration of a protein is
negatively proportional to the log of the proteins mass
What must gels be?
strong, hydrophillic, uncharge , stable and have an adjustable pore size
What would happen if there wasn't stacking gel added?
the gel would be very wide and unreadable
What is logarithmic migraion?
the moleculear weight is closer at the top than the bottom 10 and 15 have the same distance apart as 150 and 250
Electrophoresis
the movement of charged molecules toward and electrode of the opposite charge
What is fixing?
using dyes requires this to fix the protein to the gel via ethanol, methanol or acetic acid to make sure they dont leave teh fel
Autoradiography
most sensitive gel detection that uses radioisotypoes and x-ray film
Once a protein is fixed and bound to a due are they usable with other techniques?
no, normally duplicate gels are made one for staining and one for western blot
Densitometer
used to measure the intensity of bands, which would indicate a relative protein concentration compared to a known marker
Lysozyme weight
14 kDa
Gel electrophoresis
the seperation of charged molecules through a gel matrix in order to sort them by size or charge
What is the stacking gel?
5% acrylamide cast on top of resolving gel and contains wells proteins samples will be loaded into
Size of BSA
67 kDa
What is the counterion?
glycine
What kind of chemicals can break disulfide bridges?
B-ME or dithiothreitol
What is bromophenol blue?
a small, negatively charged molecule that gives the protein sample a blue color
What types of isotypes can be used?
35S, 32P, 33P, 14C and 3H
What is a loading control?
like beta-actin, can be used for normalization to account for variations in pipetting between lanes after gel is tained
What does the dominate SDS charge cause?
separation of denatured, detergent-coated polypeptide chains will be due almost exclusively to the difference in sizes of the polypeptide chains and not the difference in charges
Are oligomers broken during this process?
yes
What is silver staining
10-100x more sensitive than coomassie, offers nanogram detection where silver atoms bind tightly to proteins and produce black or purple bonds
Resolving gel
10-15% acrylamide gel undeneath the stacking gel
Charge to mass ratio
0.5
What is the SDS ratio?
1 SDS molecule per 2 amino acids
Will disulfide bridges be broken in this process of adding a sample loading buffer?
No! they must be broken with special chemicals
What is SDS PAGE?
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
What is a sample loading buffer
addition of SDS, beta-mercaptoethanol, bromophenol blue and glycerol to the protein sample
What do newer coomassie blue agents do?
allo the bands to be viewed directly during staining
Why would multiple bands show up in one lane?
because they are different modifications of the band
What does sodium dodceyl sulfate do?
binds to amino acids and coats proteins in an overwhelming negative charge
Acrylamide will only polymerize
into long chains so the cross linking agent N,N' methylene bisacrylamide is necessary to generate pores
Why is glycerol added to the sample?
it makes the samples more dense than the running buffer and ensures they sink into the wells
SDS PAGE seperates based on?
molecular weight alone
What does polyacrylamide gel do?
prevent loss of resolution caused by diffusion and vibrational and convectional disturbances and allows one to retain the seperation of different proteins after completion of eh experiment
Tetramethylethylenediamine (TEMED)
radicals induce cross linking and polymerize acrylamide and BIS
Relative mobility
ratio of distance each protein migrate to the that of tracking dye
Which gel preforms the seperation of proteins?
resolving gel
The seperation of proteins gel electrophoresis is influenced by
size of the proteins and charge of proteins at the buffer pH
More acrylamide = ___ pores and ____ migration
slower migration and smaller pores
Acrylamide
small molecule that will polymerize into chains under the right condition and the porosity of teh polymerized polyacrylamide gel can be controlled
What is pore size determined by?
the concentration of acrylamide
What part of the gel is used for tracking distance?
the top of teh gel
What happens after a sample loading buffer is added?
they are heat to near boiling to denature proteins. The detergent and heat dirsupt weak bonds
How can the bands on gels be fixed?
they can be dried in cellophane and taped to a lab notebook
What is the a common visualizing agent for protein on gel?
tightly binding dyes like coomassie brilliant blue
Disadvantages to autoradiography
time consuming and requires special certifications
The first few lanes contain
what was loaded, flowthrough and wash
