Bio 4010 Sections 4& Solutions Quiz 1. The graph below shows the result of a siz
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Bio 4010 Sections 4& Solutions Quiz 1. The graph below shows the result of a size exclusion chromatography using a standard misture of three colored molecules: blue dextran (blue, > 500 kDa), bovine hennoglobin (reddish-brown, SAS kDa·and vitamin B12 (pink. 35 kDa. The pals represent the absorbance values of the fractions in different elution time. Based on your knowledge identify which peak represents each molecule. Explain your answer. we are able to detmne tHlese pears Fraction Number 2. In your protocol, you will be asked to prepare 10x SDS-PAGE running buffer solution. To make IL of 10x running buffer solution you will dissolve Tris base (302g-121,14 MW) and glycine (144g 75,07 MW) together in 800mL of wader, add 10g of SDS and mix carefully. Chock the pHH, and complete to IL of water. Express the concentration in terms of mM for Tris base and Glycine and %w/v for SDS. Show you calculation, 3. SDS-Polyacrylamide gel clectrophoresis (SDS-PAGE) is a technique to determine molecular weight of a protcin. Following the steps of sample preparation and running the gel explain the principle of the important components of making a SDS-PAGE analysis. STEP I -PREPARING THE PROTEINS SAMPLES WITH 2X LAEMML SAMPLE BUFFER AND * (DITHIOTHREITOL) DTT. Why do you need to add DTT and Laemmli sample buffer? The sample buffer contains bromophcnol bluc, Protein after DTT STEP 2-DISCONTINUOUS SDS-PAGE SETUP. The figure illustrates the components of a discoetinious SDS-PAGE techniquc. Identify the two gels used to a polyacrylamide gel. The running buffer contains SDS (sodium dodecylsalfate). Why is SDS addod? What is the difference between the two electrophoresis techniques PAGE-Native and SDS- PAGE? The running buffer has an 8.3 pH. Why is the pll important for the SDS-PAGE irection a technique? (HINT-pl and SDS will be crucial for the separation of your proteim). How does the electrophoresis box move the proteins down the gef?Explanation / Answer
peak 1 - Blue dextran
peak 2 - hemoglobin
peak 3 - vitamin B12
Size exculsion chromatography based on principle it separate
molecules based on size. The beads are packed in the coloum.
The beads have numerous pores inside it. The small molecules enter the pores
and moves down. But larger molecules can't able to enter inside the beads
so it moves fast and elute first and smaller molecules elute last.
2. molarity = moles of solute / liters of solution
Tris
Mass = 30.2 g
volume = 1l
Molar mass = 12114 g/mol
= 30.2/12114 mol
= 0.0025
= 2.5 mmol
Molarity = 2.5/1l = 2.5 mM
Glycine Mass = 144g
volume = 1l
Molar mass = 7507 g/mol
= 114/7507 mol
= 0.015
= 15 mmol
Molarity = 15/1l = 15 mM
SDS 10g / 1000ml = 1g/100ml = 1% W/V
1. DTT is used as a protecting agent that prevents oxidation of thiol groups.
Laemmli sample buffer contains,
2-mercaptoethanol reduces the intra and inter-molecular disulfide bonds.
The SDS detergent denatures the proteins and subunits and gives each an overall negative charge so that each will separate based on size.
The bromophenol blue serves as a dye.
2. The SDS detergent denatures the proteins and subunits and gives each an overall negative charge so that each will separate based on size.
Native page no SDS use.
native PAGE the mobility depends on both the protein's charge and its size.
SDS page - SDS is added.
It separate based on size only.
3. Glycine - At low pH it is protonated and thus uncharged. At higher pH it is negatively charged. When the power goes on the glycine ions in the running buffer want to move away from the cathode (the negative electrode) so they head toward the sample and the stacking gel.
The Cl- ions move much more quickly in the electric field and they form an ion front that migrates ahead of the glycine.
4. MWM is used has a reference to identify the unknown protein molecular weight.
5. bottom of the gel.
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