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21 Cards in this Set
- Front
- Back
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Where do lipids occur? |
Lipids occur in tissues in a variety of physical forms
Complex lipids are usually constituents of membranes, where they occur in close association with proteins and polysaccharides |
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How do lipids interact? |
Lipids interact by hydrophobic and van der Waals forces and maybe by ionic bonds |
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What solvents or solvent combinations as extractants do most analysts use? |
Chloroform-methanol |
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What is contents of the signal phase Bligh/ Dyer mixture in Lipid Extraction? What is the purpose of this step? |
Chloroform/Methanol/Water (1:2:0.8 v/v) This step extracts the lipids from the cells/tissues |
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What addition of (Chloroform and Water) must be added to the Bligh/Dyer mixture to give a two-phase mixture? |
1.32 ml Chloroform and 1.32 ml Water
New ratio = 2:2:1.8 (v/v) |
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After the two-phase mixture, in Lipid Extraction, what process occurs? (i.e. extraction steps) |
First Extraction of lower phase Then add 2.64 ml Chloroform Lastly, do a second Extraction and remove the lower phase again |
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After the Extraction Steps, what is added to the two-phase Bligh/Dyer mixture and why? **What is its importance?** |
Add 5.28 ml of methanol and 3.6 ml of water, to the pooled lower phases. Gives a two-phase Bligh/Dyer mixture and is referred to as washing the lower phases **This step will clean up the lipids and is necessary for more sensitive downstream application (i.e. high pressure liquid chromatography and/or mass spectrometry** |
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Once the lipid extractions are complete, what is the ratio of chloroform/methanol that the sample is dissolved in? |
4:1 Chloroform/methanol |
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Thin Layer Chromatography |
tank must have been equilibrated for 2 hours prior to running plate
The best solvent system is the ratio 65:25:10 Chloroform: Methanol: Acetic Acid
The run takes 1 hour and 40 minutes Dry plate for 15 minutes
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Charring |
Spray plate with 10% Sulfuric Acid in 100% Ethanol Dry for 10 minutes Place on Aluminum Hot Plate (covered w/ aluminum foil--Temp. 160-180 C There is a minimal background, yet dark brown/black/purple spots for lipid species |
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What is the starting spot of the lipid extraction points called? |
The origin |
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How do you calculate the Rf value? What is the purpose of the solvent? |
Rf = x/y *x is the distance from the lipid to the top of the plate *y is the distance from the origin to the top of the plate The Solvent carries the lipid species up the plate |
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What is the principle behind the protein separation in SDS gels? |
Assumes that the proteins bind negatively charged SDS molecules along their length giving a uniform negative charge per unit of size (length) Separated based on migration through pores |
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What is Relative Mobility (Rm)? |
Calculated by dividing the distance of migration of the sample protein by the distance of migration of the tracking dye |
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Using the Rm value, how can an unknown be found? |
A graph of log molecular weight (y-axis) vs. Rm (x-axis) is constructed, and the molecular weight of known proteins is determined using the Rm of the unknown. The interpolate this onto the standard curve. |
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How many and what types of seeds are used in the electrophoretic separation lab? |
10 Homogenized seeds |
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What is the voltage used to electophorese the gel? How much time does it take for the sample to migrate to the bottom of the gel? |
100 volts 90 to 120 minutes |
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What type of dye is added to the gel? |
Bromophenol blue |
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After electrophoresis, what is the gel placed in to fix the proteins in the gel and keep them from diffusing? |
50% methanol |
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When preparing a SDS acrylamide gel, what are the steps in forming the first glass plate? |
Mix ingredients GENTLY!! (ensuring no air bubbles form)
Pour into glass plate CAREFULLY, and overlay gel with isopropanol
Wash of isopropanol w/ H2O after gel set for 15 minutes |
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What is Electrophoresis? |
Electrophoresis is the migration of charged molecules in solution in response to an electric field |
