1. SDS-PAGE & Bradford assay

2. Purpose
Understanding the principles of polyacrylamide gel electrophoresis(PAGE)
Construct standard curves and determine protein concentrations by the Bradford method

3. SDS-PAGE Theory
The movement of a charged molecule in an electric field
v = Eq/f
where v = velocity of the molecule
E = electric field in volts/cm
q = net charge on the molecule
f = frictional coefficient (depends on mass and shape of molecule

4. The Purpose of SDS-PAGE
In theory, the size and shape of a molecule
However, a variety of factors limit information about shape
But size information CAN be obtained, under the right conditions
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) is designed to give size information

5. Polyacrylamide Gels Gel of choice for protein separations
Prepared by the free radical polymerization of acrylamide and N,N’-methylene -bis-acrylamide
(a cross-linking agent)
Polymerization is controlled by an initiator-catalyst system, ammonium persulfate and N,N,N’,N’- tetramethylethylenediamine (TEMED)

6. Polyacrylamide Gels-Advantages
Good resolving power for small to medium sized proteins (best for 30,000 to 300,000)
Useful with large sample sizes
Pore sizes can be selected by varying the concentration of cross-linker
acrylamide concentration(%)
linear range of separation(kD) 15
12-43 10
16-68
7.5
36-94 5
57-212

7. The Starting Materials

8. The Polymerization Reaction
Ammonium persulfate forms radicals in solution
These radicals react with acrylamide to form acrylamide radicals
Acrylamide radicals react with acrylamide to form long polymer chains
Solutions of this polymer are viscous but do not form gels

9. Crosslinking to Form Gels
The polymers must be crosslinked to form a gel
This is accomplished by polymerizing in the presence of a “crosslinker” - N,N’-methylene-bis(acrylamide)
The product is a large network of linked acrylamide chains

10. Varying the Size of the Net
The size of the pores in the acrylamide net depends on two parameters:
the amount of acrylamide per volume
the degree of crosslinking
Other Components
Ammonium persulfate is the “initiator” of the free radical polymerization
TEMED (tetramethylethylenediamine) is a “catalyst” of the gel formation reaction, because it readily adopts a free radical form

11. Composition 30% acrylamide/bis-acrylamide solution(29:1) Neurotoxic!!
1.5 M Tris-HCl pH 8.8
: Dissolve g of Trisma base in 80 ml of dH2O then make pH 8.8 w/ conc. HCl and make up to 100 ml
10% SDS
:10 g SDS/ 100 ml dH2O
1 M Tris-HCl pH 6.8
:Dissolve 12 g of Trizma base in 60 ml of dH2O then make pH 6.8 and make up to 100 ml.
10% ammonium persulfate
: Dissolve 0.5 g of APS in dH2O up to 5 ml
SDS Running Buffer
:3.5g of Trizma base , 14.4g of glycine and 10ml of 10% SDS in dH2O up to 1000 ml
SDS sample Buffer (4X)
: 2ml of 1M Tris-HCl (pH 6.8), 0.8g of SDS, 0.75g of DTT, 250ul of 1%BPB, 7ml of 50% Glycerol

12. Composition 마지막에 넣기 Running gel (10%) Stacking gel Gel volume (ml) 104
H2O
4.0
2.7
30% acrylamide mix
3.3
0.67
1.5M Tris (pH8.8)
2.5 -
1M Tris (pH6.8)
0.5
10% SDS
0.1
0.04
10% APS
TEMED
0.004
마지막에 넣기

13. SDS-PAGE Proteins 4 % PA stacking gel “loaded” pH 6.8 into wells
glycinate<X<Cl-
% PA
resolving gel
pH 8.8
X<glycinate<Cl-
The proteins “stack up” in a narrow space
before entering the resolving gel -- leading
to more distinct bands and better resolution

14. SDS-PAGE
Glycerol in the sample buffer increases the sample density and the sample “sinks” into the well to ensure
“protein loading”
Bromphenol blue ( a small strongly
charged dye) helps guide sample loading..
…and allows you to monitor the rate of
electrophoresis. Note that it will migrate
faster than any protein

15. SDS-PAGE 1. 2. 3. 4.
Proteins are maintained in their native folded stated by a variety of molecular interactions, including disulfide bonds
Addition of mercaptoethanol reduces
disulfide bonds
Ionic detergent disrupt other interactions
and gives the protein a net negative charge
As a result, the protein is converted into a
rod-like moecule whose length is proportional
to its molecular mass - +

16. Procedure 1. SDS-PAGE 조성에 따라 각 solution을 섞는다.
(APS와 TEMED는 mixture를 붓기 전에 넣는다.)
2. Running gel을 적정 선 까지 넣고, 증류수로 채워준다.
3. Running gel이 굳고 나면, 증류수를 제거하고 stacking gel을 부어준다.
4. Comb을 꽂고 stacking gel을 굳힌다.
5. Comb을 제거하고, gel을 electrophoresis kit에 casting하고 running buffer를
채워준다.
6. size-marker와 sample을 loading하고, electrophoresis.
7. Gel running이 끝나면, caster에서 gel만 분리하여 CBB로 staining한다.
8. Destaining solution으로 destaining하여 band만 나타나게 한다.

17. Bradford Assay – Coomassie Blue
Amax of CB G-250 shifts from 465 to 595 nm
when bound to protein
dye reacts primarily with Arg
lesser extent with His, Lys, Tyr, Trp, Phe
sensitivity is mg/ml depending on circumstances
single step and few interfering substances
protein concentration extrapolated from standard curve
sample not recoverable

18. Coomassie Blue Dye lmax in absence of protein: 465 nm
when bound to protein: 595 nm (intense blue)

19. Procedure 1. Warm up the spectrophotometer. before use.
2. Dilute samples with buffer to an estimated concentration.
3. Prepare standards containing a range of 5 to 20ug/ul protein (albumin or gamma globulin is recommended) to a standard volume 800ul.
4. Add 200ul dye reagent.
5. Incubate at RT for at least 5min.
6. Measure the absorbance at 595 nm.

20. Data: Standard curve : 추세선으로 수식과 R2값을 차트에 표시할 것.
BSA (1mg/ml) final concentration
Absorbance
5 (5ug/ml)
10 (10ug/ml)
15 (15ug/ml)
20 (20ug/ml)

21. Data: Bradford Assay Sample Volume assayed (ml) Absorbance
Concentration #1 5 #2 10

22. Further Study Protein Quantification-원리, 장/단점 조사
: Lowry (copper and Folin-Ciocalteau Phenol)
: Bicinchoninic acid method
: Bradford method (Coomassie brilliant blue G250)
: Biuret method (copper and tartrate)