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Biochemistry > Experiment 8: SDS-Page > Flashcards

Experiment 8: SDS-Page Flashcards

1
Q

Electrophoresis

A

-Migration and separation of charged particles with the use of an electric field

-Separate DNA and proteins

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2
Q

Native Polyacrylamide Gel Electrophoresis (PAGE)

A

-Non-denaturing conditions
-Separation based on mass and charge

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3
Q

Sodium dodecyl sulfate (SDS) PAGE

A

-Denaturing conditions
-Separation based on mass

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4
Q

SDS Structure

A

-Hydrophobic tail inside
-Hydrophilic head outisde

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5
Q

SDS Interaction with Proteins

A

-SDS hydrophilic head attacks hydrophobic tail inside the protein.
-Uncoils protein and surrounds it with negative charge.

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6
Q

Order of samples that reach the bottom of the plate (fastest to slowest)

A
  1. Small sample (can fit in smaller pores of the gel)
  2. Medium Sample
  3. Large sample (struggle to get through the small pores of the gel)
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7
Q

SDS-PAGE Setup Components

A
  1. Container tank
  2. Black and red wires
  3. Comb
  4. Plates
  5. Sample aid guide
    6, Casting stand
  6. Casting frame
  7. Gasket
  8. Running buffer
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8
Q

Electrohporesis Chamber/Tank

A

-Holds the gel with sample, electrodes, and buffer in one container.
-Has electric current running through it to power electrophoresis.

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9
Q

Casting frame

A

Holds the two plates together

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10
Q

Casting stand

A

Stable base to hold the casting frame in an upright position

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10
Q

Comb

A

Creates wells for the samples to be placed in the gel

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11
Q

Gasket

A

Creates a seal between components and prevent leaks

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12
Q

Sample loading guide

A

A guide to know where to insert the samples

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13
Q

Polyacrylamide

A

-Stable
-Chemically inert
-Electrically neutral
-Hydrophilic
-Transparent for optical detection at wavelengths greater than 250

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14
Q

Electrode wire connection

A

-Black to black wire
-Red to red wire

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15
Q

Polyacrylamide preparation reaction

A

-Free radical polymerization of acrylamide and bis-acrylamide

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16
Q

Ammonium persulfate (APS)

A

Initiator of polymerization reaction between acryalamide and bis-acrylamide

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17
Q

Tetramethyethylenediamine (TEMED)

A

Catalyst of the polymerization reaction between acrylamide and bis-acrylamide

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18
Q

Bis-acrylamide

A

Commoner cross-linker

19
Q

Factors affecting polymerization rate

A

-Monomer concentration
-Catalyst concentration (can be adjusted)
-Temperature
-Purity of reagents

*Carefully controlled as it generates heat and lead to non-uniform pore structures if too rapid

20
Q

%T

A

Total Monomer Concentration

21
Q

%C

A

Weight percentage of cross-linker

22
Q

Optimization of Gel Pore Size

A

-Adjust %T and %C

23
Q

Smaller %T

A

Resolves bigger MW proteins

24
Q

Higher %T

A

Resolves smaller MW proteins

25
Q

Stacking gel

A

“Prepares” the samples for the electrophoresis run

26
Q

4% Stacking gel preparation

A
  1. In 100 mL beaker, pipette the ff:
    +6.1 mL distilled water
    +1.3 mL acrylamide solution
    +2.5 mL of ph 6.8 buffer
    +0.1 mL SDS solution

2.+ 50 µL ammonium persulfate solution
3.+5 µLTEMED
4. Quickly pipette solution onto casting frame until full
5. Carefully but quickly insert casting comb without introducing any air bubble

27
Q

Resolving Gel

A

Separates the different bands from each other (by maintaining negative charge of the proteins)

28
Q

10% Resolving gel preparation

A
  1. Add the ff in 100-mL beaker:
    +4.1 mL distilled water
    +3.3 mL acrylamide solution
    +2.5 mL pH 8.8 buffer
    +0.1 mL SDS solution
    • 50 µL ammonium persulfate solution
      3.+5 µLTEMED
  3. Quickly pipette the solution onto the casting frame until half of the green marking.
  4. +Tert-butanol on top to remove bubbles
  5. Allow gel to polymerize (about 15-20 minutes)
29
Q

Laemmli Sample Buffer Components

A

-Tris buffer
-SDS
-Glycerol
-Beta-mercaptoethanol
-Bromophenol blue

30
Q

Glycerol

A

Increase density than protein sample so that it really drops down

31
Q

Beta-mercaptoethanol

A

-Breaks disulfide bonds
-Only add right before the sample loading to avoid it oxidizing

32
Q

Bromophenol blue

A

-Tracks the bands throughout the run (tracking dye)

33
Q

Staining solution

A

Appearance of protein component bands

33
Q

Denaturing factors involved in SDS-PAGE

A
  1. SDS
  2. Beta-mercaptoethanol
  3. Heat
33
Q

Running buffer

A

1 x Tris-glycine pH 8.3

34
Q

Destaining Solution

A

Remove Coomassie Blue but maintain color in protein

34
Q

Staining Solution Components

A

-0.05% Coomassie Brilliant Blue Dye R250

35
Q

Destaining Solution I Components

A

50% methanol
10% glacial acetic acid

36
Q

Destaining Solution II Components

A

5% methanol
7% glacial acetic acid

37
Q

Retention Factor

A

Rf= distance to band/distance to tracking dye

38
Q

Data Analysis

A

-Graph log (MW) (y-axis) vs Rf (x-axis)
-Interpolate Rf of the unknown protein band
-Get anti-log of the resulting data

39
Q

Preparation of Casting Frames

A
  1. Take one large plate and one small plate
  2. Place them together
  3. Insert plates in a clipped stand
  4. Place onto casting frame with a rubber gasket
  5. Check for leaks and repeat if leaks are found
40
Q

Storing SDS-PAGE gels

A
  1. Remove gels from the casting stands and green frames.
  2. Keep the comb in their gel and wrap in a wet paper towel
  3. Wrap gels in plastic wrap and store at 4°C.
41
Q

Setting up the SDS-PAGE apparatus

A
  1. Short plates are facing inward
  2. Place eletrophoresis unit in the electrophoresis bath
  3. +Running buffer in inner chamber
  4. Check for leaks
  5. +Running buffer in outer chamber, not need to be completely filled.
  6. Carefully remove the comb from the gel
42
Q

Sample Preparation

A
  1. +10 µL sample
  2. +25 µL of the Laemmli sample buffer
  3. Heat over steam for 4 minutes
  4. Load samples into wells (1 sample per well) until full
  5. Plug the leads on the power supply
  6. Start power supply and run at electrophoresis at 100 V
  7. Stop electrophoresis when dye reached the end of the gel and turn off the power supply and disconnect leads from the power supply.
  8. Open the plates and cut off stacking gel
  9. Mark Lane 1 by cutting a small diagonal section on this lane
43
Q

Staining and Destaining Procedure

A
  1. Place gel on plastic tray
  2. Submerge in staining solution for 1 hour
  3. Place staining solution back in its bottle after
  4. Wash gel with tap water
  5. Submerge gel in Destaining Solution 1
  6. Dispose Destaining Solution 1
  7. Submerge gel in Destaining Solution 2
  8. Remove Destaining Solution 2 and was gel with tap water
  9. Take photo of gel against a white background.

Biochemistry (6 decks)

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