Extraction and Analysis of Proteins From Cells

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Extraction and Analysis of Proteins From Cells

• Preparation of Crude Cell Extracts

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Things to Learn in This Weeks Lab

• 1. How to extract proteins from cells.
• How to make parallel dilutions from a stock
  solution.
• How to determine protein concentration using a
  standard curve.
• 4. How to use the concentration of a dilution to
  calculate the concentration of the stock
  solution.

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Extracting Proteins From Cells
Why extract proteins from cells?
Proteins control virtually all cell activities
since they perform all kinds of functions
Cytoskeletal proteins
Enzymes
Channels, Pumps
G-proteins
Receptors
Metalloproteins
Structural Support Molecules
Storage proteins
Motor proteins
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Proteins are located in all membranes, the
cytoplasm, and the interior of all organelles.
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Proteins are amazingly diverse
charge
pH optimum
shape
size
Cofactors
solubility
This confounds purifying a specific protein.
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What kinds of protein will end up in a Crude
extract?
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Enzymes globular proteins
Hydrophilic Water soluble
substrate
enzyme
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Metalloproteins
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Channels and Pumps are Membrane Proteins
HYDROPHOBIC
HYDROPHOBIC
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How to keep a Protein Structure Intact During
Extraction
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FOUR LEVELS OF PROTEIN STRUCTURE
SECONDARY
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TERTIARY
B
A
C
D
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QUATERNARY STRUCTURE
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CARE MUST BE TAKEN TO AVOID DENATURATION
Extraction Conditions Must Maintain the Protein
in its Native Conformation and (optimally)
Preserve its Activity.
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Polypeptide A
Polypeptide A
Disulfide bond (strong)
Polypeptide B
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Polypeptide A
Polypeptide B
3. Mechanical Disruption
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Effect of pH on Protein Structure and Function
The pH determines whether or not acid or base
functional groups on a protein are charged or
uncharged.
pK value of an acid the pH at which half the
acid functional groups have given up H to the
surrounding solution, and half have not.
pH 12 very low conc. of H
pH 3 high conc. of H
pH 7 moderate conc. of H
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What is the approximate pK of this base?
pH 12
pH 3
pH 9
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Protein Extraction Buffer
PIPES Buffer
WEAK ACID
WEAK BASE
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Prevent oxidation of disulfide bonds
Dithiothreitol or Beta-mercaptoethanol (B-ME)
(reducing agents)
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Protease Inhibitors
Leupeptin
Pepstatin
EDTA
PROTEIN
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How to Maintain Structure of Extracted Proteins

• Maintain solutions at refrigerated temperatures
• Liquid nitrogen and refrigerate tools
• Resist pH changes (pH-buffered solutions)
• PIPES Buffer, pH 6.9
• Neutralize protease enzymes and cofactors
• EGTA (chelator)
• Leupeptin pepstatin (protease inhibitors)
• Avoid oxidation of sulfhydryl bonds
• DTT (reducing agent)

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Identifying and Isolating a Protein
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Beginning a Study of a Specific Protein
Example Albumin proteins maintain osmotic
balance in the blood and transport a variety of
molecules through the bloodstream.
What size(s) are albumin proteins?
How much albumin protein is in various organs?
How do you determine which cells or tissues
produce this protein?
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Steps in Protein Extraction

• 1. Select tissue liver/tripe
• 2. Pulverise tissue to get crude soluble protein
  extract centrifuge
• 3. Parallel dilution make standard purified
  (SPP) dilution series (Standard curve)
• 4. Measure absorbance of diluted extract
  calculate concentration of extract using standard
  curve

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Steps in Protein Extraction

• 1. Select tissue liver/tripe
• 2. Pulverise tissue to get crude soluble protein
  extract centrifuge
• 3. Parallel dilution make standard purified
  (SPP) dilution series (Standard curve)
• 4. Measure absorbance of diluted extract
  calculate concentration of extract using standard
  curve

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Liver Tissue Characteristics
Smooth ER
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Liver functions

• Storage of glycogen, ions, lipids, etc.
• Processing, detoxification and storage of
  molecules from intestines
• Synthesis of blood plasma proteins
• Generation of body heat
• Breakdown of RBC Bile formation

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Liver Onions Recipe

• In a small mixing bowl combine together chopped
  marjoram, thyme, parsley flakes, seasoned salt,
  prepared mustard and black pepper. Crush and mix
  all of the ingredients together to form your
  seasoning mixture. After about 40 minutes, remove
  sliced liver from milk and pat dry with paper
  towels. Coat each piece of liver first with
  seasoning mixture and then with flour. Heat
  vegetable oil in a large heavy skillet over
  medium-high heat. Fry the liver to your desired
  doneness. Remove cooked liver and place on a
  warmed plate until onions are done. Place onions
  into skillet, season with salt and pepper and
  brown, turning occasionally. Serve with steamed
  rice, vegetables and cornbread.
• http//www.soulfoodandsoutherncooking.com/liver-an
  d-onions-recipe.html

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The Omasum, tripe (third division of a
ruminants stomach)
Exterior
Interior
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Tripe
Secretory layer Muscle layer Arteries
Veins Connective tissue
d0.biggestmenu.com/.../ 20/b93d008311646a0f_m.jpg
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Steps in Protein Extraction

• 1. Select tissue liver/tripe
• 2. Pulverise tissue to get crude soluble protein
  extract centrifuge
• 3. Parallel dilution make standard purified
  (SPP) dilution series (Standard curve)
• 4. Measure absorbance of diluted extract
  calculate concentration of extract using standard
  curve

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Extract total proteins from tissues or organs to
prepare crude extracts
centrifuge
crushed cells
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Membrane Proteins will be found in Tiny Micelles
or Vesicles.
protein
protein
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To Purify Membrane Proteins
Centrifuge the crude extract
C.E.
C.E.
transfer crude extract
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Steps in Protein Extraction

• 1. Select tissue liver/tripe
• 2. Pulverise tissue to get crude soluble protein
  extract centrifuge
• 3. Parallel dilution make standard purified
  (SPP) dilution series (Standard curve)
• 4. Measure absorbance of diluted extract
  calculate concentration of extract using standard
  curve

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Quantification of protein extracts.
Stain proteins with a colored dye (Bradford
reagent).
Read absorbance of dye/protein complexes on a
spectrophotometer.
Compare to absorbances of known protein
solutions on a standard curve.
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PARALLEL DILUTIONS made from a Standard Purified
Protein Stock Solution 2mg SPP/mL
C1 2 mg/mL
V1 ?
Dilutions
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A Standard Curve
Unreliable due to scatter effects
ABS 595 nm
B
the most linear part of the curve
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Steps in Protein Extraction

• 1. Select tissue liver/tripe
• 2. Pulverise tissue to get crude soluble protein
  extract centrifuge
• 3. Parallel dilution make standard purified
  (SPP) dilution series (Standard curve)
• 4. Measure absorbance of diluted extract
  calculate concentration of extract using standard
  curve

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DETERMINING STOCK CONCENTRATION FROM A DILUTION
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A Standard Curve
0.05
C2
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Activities to come

• Quantify protein content in different tissues.
• Separate proteins by size.
• Determine presence of a specific protein.

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