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Bio 196 L > Protein Chromatography > Flashcards

Protein Chromatography Flashcards

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1
Q

chromatography

A

method to separate molecules

examples of molecules that can be separated:

  • sugars
  • amino acids
  • proteins

Separation can be based on PHYSICAL properties:

  • size
  • charge (positive or negative; hydrophobic or hydrophilic)
  • the three dimensional conformation of the molecule
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2
Q

hydrophobic

A
  • having no affinity to water
  • tend to coalesce and form droplets in water
  • does not have a charge; not polar
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3
Q

hydrophilic

A
  • Having an affinity to water

- has a charge; polar

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4
Q

size exclusion chromatography

A
  • separate on the basis of their size

- gel filtration

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5
Q

How did the size exclusion (gel filtration) chromatography work?

A
  • Mixture of proteins were added to a column containing microscopic porous beads.
  • Any molecule that is smaller than the holes in the beads enter and exit the beads at will and take a long time to travel from the top to the bottom of the column in their extended zig-zag journey.
  • Molecules that are larger than the holes in the beads ravel down the column in a direct journey very quickly since they are too large to enter the holes.
  • Since the small molecules travel slowly and the large molecules travel quickly through the column material, a method of separation is established based on size.
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6
Q

Size exclusion (gel filtration) chromatography PROCEDURE

A
  1. Obtain 12 test tubes. Label the first 10 as 1-10, and the remaining two as WASTER and BUFFER. Add 4 ml of column buffer to the tube marker buffer using transfer pipettes.
  2. Obtain one prepared column, remove the top cap and snap off the bottom end. Allow the liquid buffer to drain from the column into the tube marked WASTE.
  3. Place the column into tube 1 and carefully add 3 drops of the protein mix to the top of the column matrix with a pipette.
  4. Once the protein mix has entered the column matrix, carefully add 250 ul (.25 mL) of column buffer to the top of the column matrix. Carefully add 3ml of column buffer to the top of the column matrix. Transfer the entire column to tube 2 and collect 5 drops.
  5. After collecting 5 drops in tube 2, transfer the entire column to tube 3 and collect 5 drops again. Keep repeating this until you have 5 drops in all of your collection tubes.
  6. Examine results
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7
Q

Transfer pipettes measurements

A

1000 ul = 1 mL
750 ul = .75 mL
500 ul = .50 mL
250 ul = .25 mL

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8
Q

Size exclusion (gel filtration) chromatography RESULTS

A
  • The HEMOGLOBIN (MW= 65,000) was the molecule to exit the column first. Mixture was red-brown
  • The VITAMIN B-12 (MW- 1,350) exited last and is the smaller molecule. The mixture was dark pink.
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9
Q

denaturation

A

an extreme unfolding of proteins

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10
Q

tonicity

A

the ability of a solution surrounding a cell to cause that cell to gain or lose water

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11
Q

elute

A

to remove an absorbed substance by washing with a solvent.

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12
Q

supernatant

A

the liquid above the solid pellet after centrifuge or other process of separation

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13
Q

lysozyme

A
  • an enzyme that destroys bacterial cell walls; in mammals, it is found in sweat, tears, and saliva
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14
Q

TE buffer

A

low-salt buffer

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15
Q

binding buffer

A

high salt buffer

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16
Q

wash buffer

A

medium salt buffer

17
Q

How did the Hydrophobic Interaction Chromatography (HIC) work?

A
  • Proteins were isolated via affinity to a hydrophobic matrix
  • Increasing tonicity of the solution using HIGH salt (binding buffer) caused the protein to unfold, exposing the hydrophobic amino acids. The protein STUCK to the column matrix
  • To elute the protein we added a LOW salt buffer (TE buffer); this caused the protein to refold, hiding the hydrophobic amino acids in the interior of the protein again.
18
Q

Hydrophobic interaction chromatography (HIC) PROCEDURE

A

PART A: SAMPLE PREPARATION:

  1. Transfer 1.5 ml of bacteria overnight growth to an Eppendorf tube, labeled on top with your initials. Spin in microcentrifuge for 3 min.
  2. Carefully remove the supernatant. THIS supernatant contains some bacteria, dispose in biohazard waste.
  3. Add 250ul (.25 mL) TE buffer (low salt) to the pellet and vortex until all of the pellet is dissolved.
  4. Add 2 drops of lysozyme, mix, place at 32 degrees C for 20 min (in water bath). The lysozyme will burst open the E. coli so the protein can be removed. When the cells burst open, the protein is isolated. Solution will be viscous.
  5. Spin in the microcentrifuge for 10 min. Add 250 ul (.25 mL) of binding buffer (high salt) to a test tube for step 6.
  6. This time, SAVE the supernatant and transfer it to a tube which already has 250 ul of binding buffer. THE BINDING BUFFER IS A HIGH SALT BUFFER WHICH CAUSES THE PROTEINS TO UNFOLD EXPOSING THEIR HYDROPHOBIC RESIDUES.

PART B: PURIFICATION:

  1. Remove the bottom and the top cover from the column. Set the column in a collection tube (labeled 1) and place in the test tube rack. Let all the liquid buffer drain from the column (~3-5 minutes).
  2. Add 2 ml equilibration buffer to the column and let the solution pass through the column slowly drop by drop. Depending on how the column in packed this could occur fast or slow.
  3. Add all of the solution from PART A to the column. Carefully and gently add this solution to the top of the column matrix. Let the solution pass through slowly drop by drop. Collect all of the flow though in the collection tube labeled 1. HYDROPHOBIC PROTEINS SHOULD NOW BE BOUND TO THE COLUMN WHILE HYDROPHILLIC PROTEINS HAVE PASSED THOUGH. ELUTION IS HYDROPHILLIC
  4. Transfer the column to a new collection tube labeled 2 and add 250 ul of WASH BUFFER (medium salt) and let this solution pass through drop by drop. WASH BUFFER IS A MEDIUM SALT BUFFER WHICH SHOULD ALLOW SOME MODERATLY HYDROPHOBIC PROTEINS TO BEGIN TO RE-FOLD INTO THEIR NORMAL SHAPE. AS THEY REFOLD, THEIR HYDROPHOBIC REGIONS WILL AGAIN BE BURIED AND THUS CANNOT BIND TO THE COLUMN. DURING THIS STAGE, PROTEINS THAT ARE ONLY MODERATELY HYDROPHOBIC WILL PASS THROUGH. ELUTION IS MODERATELY HYDROPHOBIC
  5. Add 750 ul TE (low salt) to the top of the column and collect the flow through in a collection tube labeled 3. TE IS A VERY LOW SALT BUFFER, IN WHICH ALL THE PROTEINS WILL REFOLD BURYING THEIR HYDROPHOBIC RESIDUES, SO ALL PROTEINS SHOULD BE REMOVED FROM THE COLUMN. THIS FLOW THROUGH SHOULD CONTAIN ON THE MOST HYDROPHOBIC PROTEINS THAT WERE PRESENT IN THE BACTERIA. ELUTION IS HYDROPHOBIC
  6. Place tubes 1, 2, and 3 side by side and examine with a handheld UV light source. only tube 3 should show any fluorescent activity (because it contains the hydrophobic proteins- fluorescent).
19
Q

viscous

A

having a thick, sticky consistency between solid and liquid

20
Q

Hydrophobic Interaction Chromatography HIC RESULTS

A
  • During Part A we separated the protein from the bacteria using centrifuge, denaturation, lysozyme, and centrifuge again.
  • We used a BINDING BUFER (high salt) to increase tonicity and denature the protein to its primary structure and expose its hydrophobic residues.
  • Next we started PART B. We opened out column matrix and added an equilibration buffer. After the equilibration buffer passes through the column matrix we added all of PART A (the protein).
    -Because the column matrix was hydrophobic, the exposed hydrophobic amino acids stuck to the column matrix. ALL OF THE HYDROPHILLIC PROTEINS PASSED THROUGH CAUSING THE ELUTION TO BE HYDROPHILLIC
  • Next we transferred the column to new test tube and added a MEDIUM SALT (WASH BUFFER). THIS ALLOWS SOME MOERATELY HYDROPHOBC PROTEINS TO BEGIN TO REFOLD INTO THEIR NORMAL SHAPE, AS THEY REFOLD, THEY CAN NO LONGER ATTACH TO THE HYRDOPHOBIC COLUMN AND PASS THOUGH. THE ELUTION IS MODERATLY HYDROPHOBIC
  • Next we transferred the column to new test tube and added a LOW SALT BUFFER (TE BUFFER) which caused all the proteins to refold, causing them to bury their hydrophobic residues, removing them from the column. THE ELUTION IS HYDROPHOBIC.
    The bacteria was modified to include a gene from the jelly fish, aequorea victora. When the gene was transcribed and translated, a hydrophobic florescent protein was produced. Test tube 3 was hydrophobic and included this florescent protein. It glowed under UV light.
21
Q

centrifuge

A
  • a device used to separate materials based on their relative densities.
  • It uses a rotor that spins very fast (sometimes tens of thousands of times per minute)
  • must be balanced and have symmetrical density.
22
Q

hydrophobic amino acids

A
  • do not have an affinity for water

- R chains do not have a charge

23
Q

hydrophilic amino acids

A
  • have an affinity for water

- R chains have a charge

24
Q

Protein folding

A
  • primary structure is the polypeptide chains
  • secondary structure is the H bonds creating alpha helix OR beta folded sheets
  • tertiary structure is the over all 3d shape
  • quaternary structure is 2 or more folded proteins connected
  • when you denature a protein you unfold it to its primary structure
  • you refold a protein by taking away what denatured it (if possible)
25
Q

What were the reagents used to prepare the green fluorescent protein (GFP) from the bacteria and what was their purpose?

A
  • TE buffer - low salt buffer was used to dissolve the pellet created from centrifuge.
  • lysozyme- used to break down the cell walls of the bacteria in order for the protein to be isolated.
  • binding buffer- HIGH salt bugger used to denature the protein to its primary structure in order to expose its hydrophobic regions which would stick to the hydrophobic column matrix.
26
Q

What is happened to the GFP at each step of the column chromatography section?

A

Test tube 1- the GFP is broken down to its primary structure with its hydrophobic regions sticking to the column matrix.
Test tube 2- The medium buffer (wash buffer) causes the moderately hydrophobic proteins to begin to refold. When the moderately hydrophobic sections go back to the center they can no longer attached to the hydrophobic column and elute into the test tube.
Test tube 3- A low buffer (TE BUFFER) causes all the hydrophobic proteins to refold into the test tube. The GFP is hydrophobic and is released from the column matrix and is now in the test tube.

27
Q

Proper way to balance a centrifuge

A

Centrifuges should be balanced by having symmetrical density by adding tubes of equal weight on opposite sides of the rotor.

Bio 196 L (9 decks)

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