Lab 3 - SDS-PAGE

Question 1

What are the tasks of this lab?

Answer 1

• Prepare resolving and stacking gel
• Protein sample preparation
• Load and run SDS-PAGE gel
• Stain (Coomassie Brilliant Blue)
• De-staining of gel
• Gel imaging

Question 2

What does the electrophoresis of proteins using polyacrylamide gel allow for?

Answer 2

The separation of proteins into their individual subunits

Question 3

In this lab, what strong anionic detergent will be used?

Answer 3

Sodium dodecyl sulfate (SDS)

Question 4

What does using a combination of SDS, reduction agents, and heat do?

Answer 4

Dissociates and denatures the proteins before loading them into a gel

Question 5

What will the amino acids contained within our proteins bind to?

Answer 5

The SDS and will become negative charged

Question 6

How does SDS denature proteins?

Answer 6

Minimizes differences in molecular form by eliminating the tertiary and secondary structures:
- “Wraps around” the polypeptide backbone binding to the protein in a mass ratio of 1:4:1
- Disrupts hydrogen bonds
- Blocks hydrophobic interactions
- Substantially unfolds the protein molecules

Question 7

When can the proteins be totally unfolded and how?

Answer 7

When a reducing agent such as dithiothreitol (DTT) is employed, which reduces disulphide bonds

Question 8

What is essentially a linear function of peptide chain length?

Answer 8

Molecular weight

Question 9

How does the size of the protein complex determine how it migrates through the matrix?

Answer 9

In sieving gels, the proteins separate by molecular weight, therefore, the larger the complex, the more slowly it will migrate

Question 10

What does the separation ability of an SDS-PAGE gel depend on?

Answer 10

The concentration of the polyacrylamide used to make the gel and the amount of cross-linking

Question 11

What is the gel matrix comprised of?

Answer 11

Chains of polyacrylamide cross-linked by a bifunctional agent such as bis-acrylamide (N,N’-methylene-bis-acrylamide

Question 12

What does the addition of bi-acrylamide add to the gel?

Answer 12

Add rigidity and tensile strength and forms pores which allow the SDS:protein complex to pass through

Question 13

As the bisacrylamide:acrylamide ratio increases, how does the size of the pore change?

Answer 13

Decreases

Question 14

What does a ratio of 1:29 SDS-PAGE gels allow for?

Answer 14

The dissociation of peptides that differ in size by as little as 3%

Question 15

What will the SDS-PAGE in this lab be carried out in?

Answer 15

A discontinuous buffer system in which the bugger in the reservoirs and the sample has a pH and ionic strength different than the buffer used to case the gel

Question 16

What is the pH and ionic strength of the stacking gel?

Answer 16

pH 6.8 and 0.5M Tris-HCl

Question 17

What is the pH and ionic strength of the resolving gel?

Answer 17

pH 8.82 and 1.5M Tris-HCl

Question 18

What is the pH and ionic strength of the resolving gel?

Answer 18

pH 8.82 and 1.5M Tris-HCl

Question 19

What is the pH of the upper and lower buffer reservoirs?

Answer 19

Tri-glycine pH 8.3

Question 20

What happens after the protein migrates through the stacking gel of higher porosity?

Answer 20

The protein:SDS complex is deposited in a very thin zone (or stack) on the surface of the resolving del

Question 21

What does the discontinuous buffer system effectively concentrate the protein into?

Answer 21

A very small volume allowing for increased resolution in the SDS-PAGE gel

Question 22

Within a gel, what linear relationship exists?

Answer 22

One between the logarithm of the molecular weight of an SDS-denatured polypeptide, and its Rf

Question 23

How is the Rf value calculated?

Answer 23

As the ratio of the distance migrated by the molecule to that migrated by a marker from dye-front

Question 24

What is a simple way to determine relative molecular weight by electrophoresis (Mr)?

Answer 24

Plot a standard curve of distance migrated vs. log^10MW for known samples, and read off the log Mr of the sample after measuring distance migrated on the same gel

Question 25

How are proteins that have been separated by polyacrylamide gel electrophoresis typically detected?

Answer 25

By staining - this can be completed by using either Coomassie Brilliant Blue R250 or silver staining

Question 26

What kind of dye is Coomassie Brilliant Blue R250 and what does it allow for?

Answer 26

Aminotriarylmethane dye which forms strong complexes and stoichiometrically binds with proteins, but does not bind tightly to the SDS-PAGE gel, therefore you can visualize and estimate the relative abundance of the proteins of interest as discrete bands within the translucent matrix of the gel following destaining

Question 27

What can proteins separated by SDS-PAGE be fixed by?

Answer 27

Using methanol or ethanol:glacial acetic acid and stained with Coomassie

Question 28

What is the uptake of the dye proportional to and what does it mean?

Answer 28

The amount of protein, therefore, any unincorporated dye that is not bound to proteins can be allowed to diffuse from the gel in a de-staining step

Question 29

What is the procedure for gel cassette preparation?

Answer 29

1) Ensure plates are clean - if needed, spray with 70% ethanol and wipe with kim wipe
2) Place a Short Plate on top of the Spacer Plate, with 1 mm spacers facing inwards
3) Slide two plates into Casting Fram (with cams pushed inwards), keeping the Short Plate placing front - ensure both plates are flush at the bottom on a level surface
4) Lock the pressure cams by pushing outwards to secure the glass plates
5) Engage the spring loaded level and place the gel cassette assembly on the gray casting stand gasket
6) Full the inside of the plates with ddH2O to check for leaks and then after a few minutes, drain it out
7) In a screw-cap tube, mix 5 mL of resolving gel but do NOT add the ammonium persulphate (APS) or TEMED until you are ready to cast your gel
9) Pipette the solution down the spacer into each sandwich to a level about 4 cm from the top
10) Gently apply approx. 0.5 mL of water to the resolving gel and pour off once gel has fully polymerized
11) Mix stacking gel and fill into sandwich and insert a comb
12) Allow gel to polymerize

Question 30

What is the recipe for the resolving gel (10%)?

Answer 30

• 2400 uL ddH2O
• 1250 uL of 40% acrylamide/bisacrylamide
• 12500 uL gel buffer (1.5 M Tris-HCl pH 8.8)
• 50 uL SDS (10% w/v)
• 50 uL ammonium persulphate
• 4 uL TEMED

Question 31

What is the recipe for the stacking gel (5%)?

Answer 31

• 1750 uL ddH2O
• 300 uL of 40% acrylamide/bisacrylamide
• 400 uL gel buffer (0.5 M Tris-HCl pH 6.8)
• 25 uL SDS (10% w/v)
• 25 uL ammonium persulphate
• 4 uL TEMED

Question 32

What is the procedure for loading the gels?

Answer 32

1) Remove Gel Cassette Sandwich from Casting Frame and place into the Electrode Assembly with the Short Plate facing inward
2) Slide the Gel Cassette Sandwiches and Electrode Assembly into the clamping frame
3) Press down the Electrode Assembly while closing the two cam levers on the Clamping Frame
4) Lower the Inner Chamber into the Mini Tank
5) Fill inner chamber with tank buffer (1xSDS PAGE running buffer), then the outer chambers
6) Adjust height of tank buffer until sandwiches are fully immersed in buffer
7) Remove comb from gel and rinse each well with tank buffer

Question 33

What is the procedure for preparing protein samples?

Answer 33

1) Thoroughly resuspend “Total Cell Culture - Uninduced” and “Total Cell Culture - Induced” in 100 uL of 1xPBS
2) Prepare all samples by combining 20 uL with 4 uL of 6xSDS loading dye
3) Place tubes in a 95 degrees C heating block for 5 minutes
4) Slowly add 20 uL of each sample in each well and 3 uL of 6xSDS loading dye to empty wells
5) Put the lid on the gel unit and connect it to the Power Supply
5) Turn on and adjust voltage to 150 V and let it run until the tracking dye reaches the bottom of the gel

Question 34

What is the procedure for staining?

Answer 34

1) Immerse the gel in at least 5 times the staining solution and cover
2) Place the container in the microwave and heat for 10 seconds
3) Close the lid and place on a slowly rotating platform for 15-30 mins to cool and finish protein staining
4) Remove Coomassie stain and place back into its 50 mL centrifuge tube
5) Wash the gel in milliQ H2O twice to remove stain before adding de-stain solution
6) Add 25 mL of an ethanol:acetic solution labeled “destain” to your gel containing container and place on the rotating platform
7) Leave gel to destain for 15 mins and then discard destain solution
8) Repeat destaining step one or two more times with fresh destain
9) At the end of the final destaining step, destain with water
10) Document the gel using the Imaging system

Question 35

What are the required lab results?

Answer 35

1) Electronic image of your Coomassie-stained gel
2) Determine the relative molecular weight of His6-PTEN using the graph generated from the relative mobility from your gel image of the standard molecular weight markers