SDS PAGE

Question 1

(SDS-PAGE) meaning

Answer 1

Sodium dodecyl sulfate - polyacrylamide gel electrophoresis

Question 2

→ A type of gel electrophoresis used to separate protein molecules by loading the sample into a gel and making the protein molecules move through the gel
→ This allows us to confirm the presence of a specific protein (different proteins form different bands), and determine the quantity of the target protein (based on intensity/thickness of bands) in the sample

Answer 2

Sodium dodecyl sulfate - polyacrylamide gel electrophoresis

Question 3

SDS-PAGE
→ Gel is stained with ________________ after the electrophoresis
→ Protein bands can be seen as blue bands under ordinary room light

Answer 3

Coomassie Brilliant Blue R-250

Question 4

→ Experimental technique that separates charged molecules (Protein, DNA, RNA) based on size, charge, and conformation/shape
→ Uses an external power supply to establish an electric field that drives the movement of the molecules through a gel/matrix

Answer 4

Gel electrophoresis

Question 5

negative electrode

Answer 5

Cathode

Question 6

appear at the 2 ends of the gel during electrophoresis are the H2 gas and O2 gas produced by the reduction and oxidation reactions

Answer 6

Bubbles

Question 7

positive electrode

Answer 7

Anode

Question 8

SDS-polyacrylamide gel electrophoresis
→ Separates proteins based on _________

→ Because proteins have different amino acid compositions…

o Proteins can have different overall charges - Variable side chains (R group) of amino acids

o Proteins form different shapes - Chemical interactions between atoms of the different amino acids

Answer 8

size (number of amino acids)

Question 9

protein sample is mixed with

Answer 9

SDS
DTT
Heat

Question 10

→ SDS-PAGE removes variation in ___________ so that only protein size affects migration of proteins in the gel

Answer 10

protein shape/conformation and net charge

Question 11

Proteins in their natural state
these 3 factors will affect movement of proteins through the gel

Answer 11

Have different sizes
Have different shapes
Have different net charges

Question 12

Proteins during SDS- PAGE:
* Have different sizes…
* BUT

Answer 12

all unfolded AND all have negative charge

Question 13

→ Amphipathic detergent - Has polar and non-polar regions
* Denatures (unfolds and linearizes) proteins by disrupting the chemical bonds
。 Gives different proteins uniform net negative charge

Answer 13

Sodium dodecyl sulfate (SDS)

Question 14

protein sample is mixed with this and SDS to Specifically disrupts disulfide linkages

(Reducing agent)

Answer 14

Beta-mercaptoethanol / Dithiothreitol (DTT)

Question 15

SDS is also added to the gel to

Answer 15

maintain denaturation of proteins

Question 16

protein sample has this applied to solution with this and SDS and DTT to disrupt the chemical bonds

Answer 16

Heat

Question 17

1 sds molecule to how many amino acids

Answer 17

2

Question 18

polyacrylamide gel Made by co-polymerization of

Answer 18

acrylamide and bis-acrylamide

Question 19

SDS-PAGE uses how many layers of gel to ensure formation of thick, concentrated protein bands

Answer 19

2

Question 20

Catalysts for cross linking Acrylamide monomer
and Bis-acrylamide monomer

Answer 20

Ammonium persulfate (APS)
Tetramethylethylenediamine (TEMED)

Question 21

gel layer in SDS
upper layer
→ Larger pore size
→ Lower acrylamide/bis-acrylamide content Ensures entry of all proteins into the resolving gel at the same time

Answer 21

Stacking gel

Question 22

gel layer in SDS
lower layer
→ Smaller pore size
→ Higher acrylamide/bis-acrylamide content Enables separation of proteins based on molecular weight

Answer 22

Resolving / Separating gel

Question 23

The polyacrylamide gel
→ Sample recipe for stacking gel and resolving/separating gel

Answer 23

Reagent
Resolving gel
Stacking gel
Deionized water
30% acrylamide:bis-acrylamide (29:1)
Tris-HCl, 0.4% SDS pH 8.8
Tris-HCl, 0.4% SDS pH 6.8
10% ammonium persulfate (catalyst) TEMED (catalyst)

Question 24

Tris-HCl, 0.4% SDS pH

Answer 24

8.8

Question 25

Tris-HCl, 0.4% SDS pH

Answer 25

6.8

Question 26

30% acrylamide:bis-acrylamide ratio

Answer 26

29:1

Question 27

How to ensure stacking of proteins in the stacking gel?

Answer 27

o 2 gel layers, different pore size and buffer pH level o 3rd buffer with glycine

Question 28

→ SDS-PAGE uses what kinds of buffer system

Answer 28

discontinuous buffer system

Question 29

→ Charge of glycine changes depending on the pH low neutral high

Answer 29

low = + neutral = neutral high = negative

Question 30

Other components of SDS-PAGE → Sample solution (Protein sample + treatment buffer) contains:

Answer 30

- The protein sample - SDS - Beta-mercaptoethanol (Reducing agent) - Tris-HCl buffer, pH 6.8 → same as stacking gel buffer -Glycerol - denser than water, Sample sinks because of glycerol

Question 31

loading/tracking dye used to allow us to track the progress of the electrophoresis

Answer 31

Bromophenol Blue

Question 32

A resolved dye front, where the dye has migrated to the bottom of the resolving gel, indicates

Answer 32

that the electrophoresis is complete.

Question 33

AFTER gel electrophoresis → Visualization of protein bands

Answer 33

1) Staining with Coomassie Brilliant 2) WESTERN BLOTTING or IMMUNOBLOTTING

Question 34

→ Before loading samples in the SDS-PAGE gel, we should determine the ____ for each sample that contains our target total amount of proteins

Answer 34

specific volume

Question 35

Importance of loading in the gel volumes of the different samples that contain the same total amount of proteins

Answer 35

it is possible that the decrease or increase in band intensity is not caused by treatment but is the result of loading unequal total amounts of proteins for the different samples

Question 36

Problems with SDS-PAGE gel results → Typically caused by loading a large volume of sample, or samples that have very high concentration → Can also result from using very high voltage during electrophoresis

Answer 36

→ Smearing

Question 37

Problems with SDS-PAGE gel results → Likely caused by loading a small volume of the sample, or samples with very low concentration

Answer 37

→ Bands are too faint

Question 38

Problems with SDS-PAGE gel results → Consequence of prematurely ending the electrophoresis

Answer 38

→ Bands appearing only in the upper portion of the gel

Question 39

Problems with SDS-PAGE gel results → Caused by running the gel too long → Dye molecules have exited the gel; thus, there is the possibility that smaller proteins in the sample also already moved out of the gel

Answer 39

→ No dye front

Question 40

Problems with SDS-PAGE gel results →Typically caused by uneven surface of the resolving gel after it solidified → Can also be caused by protein overloading or the presence of impurities (e.g., cellular debris, unpolymerized acrylamide)

Answer 40

+ Streaks

Question 41

why do streaks happen with uneven surface of resolving gel

Answer 41

* Because of the uneven surface, proteins in the sample become more concentrated in some areas upon entering the resolving gel, causing their aggregation/precipitation * Aggregated/Precipitated proteins may redissolve as they move through the resolving gel, but they no longer migrate at the same time as other proteins of the same size

Question 42

Problems with SDS-PAGE gel results → Typically caused by uneven distribution of heat generated by the electric current → Typically caused by tank buffer leaking out of the inner chamber

Answer 42

→ Crescent/Smiling/Frowning effect (Uneven sample migration)

Question 43

Agarose Gel Electrophoresis (AGE) vs. Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) → In AGE, SDS (w/c makes proteins uniformly negatively charged) is not used since

Answer 43

DNA is already negatively charged

Question 44

Agarose Gel Electrophoresis (AGE) → is used to make the gel o Allows formation of larger pores for DNA as DNA molecules are bigger than proteins

Answer 44

Agarose

Question 45

Agarose Gel Electrophoresis (AGE) → gel is in a horizontal position, indicating that (2 points)

Answer 45

。 No two gel layers 。 Agarose gel formation is not inhibited by oxygen

Question 46

Agarose Gel Electrophoresis (AGE) vs. Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) → SDS-PAGE gel (for protein electrophoresis) is made by

Answer 46

polymerizing acrylamide and bis-acrylamide

Question 47

→ SDS-PAGE gel (for protein electrophoresis) is made by polymerizing acrylamide and bis-acrylamide. The polymerization of these 2 monomers is inhibited by

Answer 47

oxygen

Question 48

Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) → gel is made by polymerizing acrylamide and bis-acrylamide. The polymerization of these 2 monomers is inhibited by oxygen. Therefore, protein gel is made by pouring the solution into a narrow space between __________ to minimize exposure to air

Answer 48

2 glass plates

Question 49

Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) → In addition, protein gel has 2 layers (an upper layer and a lower layer) to ensure formation of

Answer 49

thick, concentrated protein bands

Question 50

Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) → Making a continuous 2-layer gel while also preventing exposure of gel solution to oxygen is easily done in what direction

Answer 50

vertically

Question 51

After loading the protein samples into the gel, put the lid of the tank (black to black, and red to red) and turn on the power supply. Run the gel at __________ while the samples are still in the stacking gel. Increase to ________ when the samples reach the top of the running/resolving gel

Answer 51

150 V 300 V

Question 52

After running samples, the steps that follow should be:

Answer 52

Remove the gel cassette from the tank. Immerse the gel cassette in deionized distilled water separate the gel from the glass plates with the use of a spatula/scraper. Remove the water. Soak the gels in the staining solution (Coomassie Brilliant Blue R-250). Leave them on the shaker for 24 hrs. Protein ladder

Question 53

Protein ladder that we used for SDS-PAGE

Answer 53

All blue Precision plus,protein standard

Question 54

running tank buffer

Answer 54

tris glycine 8.3

Question 55

gel buffer

Answer 55

tris HCL 8.8

Question 56

more bis acrylamide for resolving or stacking gel?

Answer 56

resolving

Question 57

buffer and ph for resolving gel

Answer 57

tris HCL 8.8

Question 58

buffer and ph for stacking gel

Answer 58

tris HCL 6.8