Lab 3 Membrane structure and function. 1. Investigate effects of stressful experimental treatments...
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Transcript of Lab 3 Membrane structure and function. 1. Investigate effects of stressful experimental treatments...
Lab 3 Membrane structure and function
1. Investigate effects of stressful experimental treatments on living membranes2. Investigate concepts about membrane structure3. Learn basic principles of spectrophotometry and gain experience using spectrophotometer
Objectives
The Membrane: Overview
The Membrane: Overview
The Membrane: Overview
Phospholipids
Phospholipids contain hydrophobic and hydrophilic regions (amphipathic)
The heads are polar which makes them hydrophilic (water loving)
The tails are non-polar which makes them hydrophobic (water hating)
This molecular structure is what allows phospholipids to form membranes
Phospholipids form a bilayer
Membrane is embedded with proteins
Membrane contains many proteins
Peripheral proteins are bound to the surface of the membrane
Integral proteins penetrate the hydrophobic core
These proteins provide a wide variety of functions for the cell
Fluid mosaic model The membrane is FLUID
Lateral movement of phospholipids is rapid
Fluidity of the membrane is important to its function
Fluidity changes with temperature
Fluidity depends on the composition of the membrane Ex: some fish live in
extremely cold environments. How do they keep their membranes fluid?
The membrane is a mosaic: many different proteins
Membrane permeability: “Are you on the guest list?” Plasma membranes are selectively permeable Permeable to non-polar molecules
hydrophobic molecules that can enter the lipid bilayer
Ex. O2, CO2
Non permeable to polar molecules (charged molecules) hydrophilic therefore cannot enter the lipid bilayer
and remain in the aqueous environment. Ex. Na+, glucose, amino acids. (there are, however, mechanisms the cell has
developed to allow import/export) what do you think the transmembrane proteins do?
Hydrophilic VS Hydrophobic
Hydrophilic- Polar (charged)- Water loving- H-bonds to H2O
Hydrophobic- Non-polar (non
charged)- Water fearing- Binds to other non-
polar molecules
C – H2.5 2.1
(close - non-polar)
O
H H2.1
3.5
Solvents used today
Methanol 2-propanol
(Ratio of polar : non-polar groups)
Polar Non-Polar
Size
SUMMARY:
Polar molecules are hydrophilic will stay in the water (H-bonds with water) and not enter the lipid bilayer of the membrane Do we expect them to cause damage to a cell
membrane? Non-polar molecules are hydrophobic therefore
will enter the lipid bilayer Do we expect them to cause damage to a cell
membrane? Does the size of a non-polar molecule influence
the extent of damage?
Today’s lab
Investigate how different temperatures and solvents can cause membrane damage
Beta vulgaris
Temperature stressOrganic solvent stress
Measure betacyanin leakage as a wayto quantify membrane damage
Betacyanin: red pigment
• Betacyanin is found in vacuole (enclosed in membrane)
• If vacuole membrane is damaged, betacyaninwill leak out (red pigment)
• How can we measure this?
Betacyanin
Spectrophotometer
525nm(the wavelength absorbed by betacyanin)
• Betacyanin absorbs light at a wavelength of 525nm
• The spec will be set to shine 525nm light on your tubes
• the amount of light being absorbed will be measured.
• The more betacyanin in the tube, the ___?___ absorbance reading.
Spec 20s
Follow the instructions in Appendix F
Betacyanin absorbs light maximally at 525nm, so you need to set the Spec to shine light of 525nm. Make sure filter lever is turned to the right range
Experimental design
Hypotheses Specific, logical
Independent variable This is the part that you are controlling ________ (Temperature)__________
Dependent variable This is the part that you measure _________ (absorbance)___________
Controls Best control is the removal of the
independent variable (ie: the controls for this experiment are room temp OR water)
Hypothesis:
A good hypothesis must: explain how or why: provide a mechanism. be compatible with and based upon the existing
body of evidence. link an effect to a variable. state the expected effect. be testable. have the potential to be refuted.
Hypothesis: make your own!“I hypothesize that …”
“The rationale for the hypothesis is…”
Remember
You will find that certain temperatures and solvents will damage the membrane more than others. MAKE SURE YOU CAN EXPLAIN WHY certain temps/solvents damage the membrane
What is happening to: the phospholipids? the proteins?
Why does this result in betacyanin leakage?
Graphs
Temp treatment is a line graph because the data is continuous Independent variable on the X axis Dependent variable on the Y
Solvent treatment is a bar graph Follow guidelines in Appendix Graph should be full page Include your data AND class data