Lab Presentation

Question 1

What are the steps of protein extraction and purification?

Answer 1

1. find source of protein by mechanical pertuburbation to break open cell wall and extract cytosolic proteins
2. Protein extraction continues with centrifugation which separates large impurities
3. Dialysis which allows for selection of target solution conditions for protein
4. affinity chromatography involves retardation of molecules with respect to solvent that progresses through material

Question 2

proteins ligand

Answer 2

ligand of protein can be attached to an insoluable matrix in conditions that favor reversible binding. to elute the protein the binding of ligand must be broken

Question 3

Immobolized Metal Affinity Chromatography

Answer 3

1. relies on formation of weak coordinate bonds between immobilized metal ions and amino acid residues of histidies. 2. The binding buffer conditions needs to ensure protein interacts with resin.
2. Used to wash unbound substances from the column without eluting the target protein

Question 4

How do you elute the protein from the column?

Answer 4

Imidazole binds stronger than LDH A protein to resin. This elution phase buffer break the interactions between the target protein and the resin

Question 5

How do you visualize purification and concentration of LDH A?

Answer 5

gel electrophoresis-seperates through mass to charge ratio

Question 6

What does LDH bind?

Answer 6

NAD+

It reaction is NAD+ +Lactutate=> NADH + pyruvate

Question 7

Histidine binding

Answer 7

LDH A enzyme has histidine primary structure residues, therefore histidine has a high affinity for nickel that allows it to bind the column and be eluted with imidazole.

Question 8

Imidazole how does it work?

Answer 8

similar molecular structure to histidine and competes with histidine for binding to the metal-charged resin and is used for elution of LDH A with this column

Question 9

How do you wash with imidazole?

Answer 9

10mM Imidazole-Low concentration of imidazole to binding and wash buffers to interfere with weak binding of other proteins and elute proteins that weakly bind

Question 10

How much imidazole to elute LDH A?

Answer 10

LDH A with histidine are eluted with higher concentrations of imidazole

Question 11

How do you detect LDH activity and quantify the purification? What wavelength?

Answer 11

spectrophotometrically at 340 nm by following the oxidation of NADH with pyruvate or reduction of NAD+ with lactate

Question 12

How does the Ni2+ work in terms of binding to LDH A?

Answer 12

beaded agarose or magnetic particles with chelating groups immobilize Ni2+ and function as ligands for binding and purification of LDH A

Question 13

What did the metal ion and histidine do?

Answer 13

LDH A adhere to NI2+-His resin and LDH A eluted in 100 mM elution of imidazole

Question 14

What type of muscle did we use?

Answer 14

Chicken Gallus gallus muscle cells

Question 15

Why did we purify LDH A through Ni2+-His affinity chromatography?

Answer 15

1. non-denaturing selective precipitation of LDH A through high salt allows the preservation of protein structure, producing an active enzyme who’s kinetics can be characterized
2. Natural poly-histidine tail of LDH A makes it susceptible to binding of nickel resin with imidazole elution

Question 16

What are the 6 main skills and concepts?

Answer 16

1. Extraction of muscle tissue
2. Centrifugation
3. salting out
4. dialysis and buffer exchange
5. concentration determination
6. activity assay

Question 17

Explain extraction of muscle tissue.

Answer 17

Begin with a simple blender-homogenization of muscle tissue in a buffered solution. Keep the tissue cold so that blender can heat the sample rapidly. LDH is susceptible to degradation by thermal denaturation, and proteases also present in the tissue will act more aggressively at higher temperatures

Question 18

Explain centrifugation

Answer 18

Fundmental technique in protein purification to solid material from aqueous part of sample. We use centrifugal force to accomplish this by loading out samples in a centrifuge and spinning them at high speed. Solid material will pellet at the bottom and the homogeneous liquid that remains as the supe. We had it in the supe so we decanted and left as much of the pellet behind as possible. Remove the supe by pipetting to avoid disturbing the pellet.

Question 19

What is the first step of protein purification ?

Answer 19

Separate insoluble cellular material from dissolved contents of cell which is the first step of protein purification.

Question 20

What is salting out?

Answer 20

second step of protein purification is non-denaturing precipitation of protein from solution. Precipitate LDH to separate it from soluble proteins to get it to become solid. The protein is forced out of solution by adding larger amounts of salt.

Question 21

What is the point of purification?

Answer 21

To generate an active enzyme whose kinetics we can characterize

Question 22

What was used as the salt for salting out step? How much does LDH A precipitate at?

Answer 22

Ammonium sulfate. LDH precipitates at 0.39 g/mL or 60% that is the salt’s maximum solubility.

Question 23

What is the molecular interactions of salting out?

Answer 23

By adding a large concentration of charged salt particles, all the water electrostatic interactions are taken up by the dissolved salt, leaving none to solvate the protein. The protein falls out of solution of its charged because it cannot interact with the water due to the salt taking up all the interactions with water. Concentration of salt to precipitate LDH A depends on sequence and structure of protein, such as arrangement of exposed polar and charged groups.

Question 24

How do we add the salt?

Answer 24

Slowly to ensure that its local concentration does not exceed that required to precipitate LDH because precipitating other proteins doesn’t help with purification effort.

Question 25

Explain dialysis and buffer exchange.

Answer 25

Dialysis removes the salts and other small dissolved molecules that interfere with chromatography. This is done through cellulose-based tubing with pores, which allow molecules of defined size to freely pass in and out. Pore sizes are selected so LDH can retain inside the membrane and all molecules of lower molecular weight will flow out. Concentration of small molecule solutes will reach an equilibrium concentration equal to dilution of dialyzed volume to total volume of dialystae.

Question 26

Explain concentration determination.

Answer 26

BCA assay is used to determine concentration so we saved 300 micro liters of samples from each purification step.

Question 27

How does BCA assay work?

Answer 27

Need a dilution table to generate a standard curve. Used a dilution equation. Use bovine serum albumin at a known concentration. It turns the solution purple and the colors intensity depends on protein concentration. Compare absorbance of unknown to standard curve and estimate the concentration of the unknown.

Question 28

How does the dilution of BCA work?

Answer 28

Dilution is done by making 6 standard dilutions form the table. It was more diluted as you go from 1 to 6. Water ratio increases compared to BSA stock solution. Then once that was done, to a new set of tubes we placed 25 microliters of each of the diluted standards. Label tubes of unknown protein samples and all 25 microliters of unknown protein samples to new tubes. Need a blank with 25 microliters of water. After all that is done, add 1 mL of BCA assay solution to every tube. Incubate for 30 minutes at 37 degrees C. Run assay on specific nm and plot.

Question 29

How is the enzyme activity calculated?

Answer 29

From the slope of the line

Question 30

How do you use the BSA standard curve once its generated?

Answer 30

Concentration is plotted against their absorbance. Assay determine unknown concentrations of lysate, salt-out supe, post dialysis, flow through, and affinity column peak. Concentrations found using slope of BSA standard curve and plug in absorbance for y value and solve for x. Based on this, the purification table was generated.

Question 31

What is activity assay?

Answer 31

Use a spectrophotometric assay to determine the activity of LDH. This enzyme binds to NAD+ and converts lactate to pyruvate (oxidation) through the reduction of NAD+ to NADH. The reduced form of this coenzyme has a strong absorbance at 340 nm, while the oxidized form is transparent at this wavelength.

Question 32

How does the absorbance of LDH change?

Answer 32

When enzyme is added to mixture of lactate and NAD+ we will observe a change in the absorbance as NAD+ is reduced. This absorbance will then taper off as the concentration of reactants and products reach equilibrium. The changed absorbance is used to measure presence or absence of protein.

Question 33

How do we quantify enzyme kinetics and calculate LDH activity?

Answer 33

Through Beer-Lambert’s Law with the extinction coefficient of NADH (6220 M-1cm-1).

1. use the slope (change in absorbance per minute) and divide it by the extinction coefficient of NADH. You get A which is change in absorbance per minute.
2. Calculate enzyme activity by converting it to micromoles and divide by enzyme volume of NADH produced
3. This gives you enzyme activity in the end in micromole/min*mL
4. Also don’t forget to multiple by dilution factor if assay is diluted (x10 or x100)

Question 34

What is the molecular weight of LDH A?

Answer 34

140 KDa

Question 35

When did I get lysate?

Answer 35

After the tissue was extracted, I decanted the lysate and filtered it through the cheesecloth into a graduated cylinder. I did not disturb the pellet in the centrifuge tube.

Question 36

How did I get salt out supe?

Answer 36

Based on volume of lysate, measure 0.39 g*ML-1 ammonium sulfate. Stir bar was added to lysate in a smaller beaker and this smaller beaker was placed in a larger beaker filled with ice on the stir plate. Avoid foaming and add ammonium sulfate slowly, no more than 1 g at a time. Wait for the salt to dissolve completely before adding more. This ensures the concentration of ammonium sulfate is never so high that all the proteins precipitate. 15-30 mins. Gradually decant (pour from one container into another) and centrifuge at 4 C for 30 mins at 18,000 RPM. Decant the supernatant into a Falcon tube and refrigerate and protein should be in the pellet. 300 uL of supernanant was labeled as salt out supe

Question 37

How did I get resuspended pellet?

Answer 37

2L of resuspension buffer for dialysis of protein was made. This was done one ice and pellet was dissolved in 3 mL of resuspension buffer. Dialysis bad was made by soaking 15 cm length of dialysis tubing in buffer for 5 minutes. Fold and clamp one end and leave enough tubing to fill with your resuspension mixture and clamp the end. Resuspension mixture was pipetted into dialysis tubing. 1 mL resuspension buffer was rinsed from the centrifuge tube and was pipetted into the dialysis tubing. 300 uL of resuspension mixture from dialysis tubing was removed and placed in microcentrifuge. This is resuspended pellet

Question 38

How did I get post-dialysis?

Answer 38

The dialysis bad was placed in a beaker and covered with plastic wrap. Dialyzed at 4 C overnight. After, 300 uL of dialyzed sample in micro centrifuge tube and labeled as post-dialysis.

Question 39

What was used to get resuspended pellet and post dialysis?

Answer 39

Resuspension and Solvent Exchange buffer: 10 mM Tris HCL, pH 8.6, 1mM meta-mercaptoethanol, 1 mM PMSF

Question 40

How did I do activity assay?

Answer 40

Spectrometer is blanked with water. 1 mL cuvettes with 1 mL assay buffer. Place one in spectrometer at 340 nm. 10 fold dilutions may be made of each sample. Use resuspension buffer to dilute and not water. Samples were watched for absorbance change. Found appropriate enzyme amount with correct dilution and enzyme volume to use for optimal changes in absorbance.