SDS-PAGE & Bradford assay

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

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

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

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)

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

The Starting Materials

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

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

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

Composition 30% acrylamide/bis-acrylamide solution(29:1) Neurotoxic!! 1.5 M Tris-HCl pH 8.8 : Dissolve 18.15 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

Composition 마지막에 넣기 Running gel (10%) Stacking gel Gel volume (ml) 10 4 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

SDS-PAGE Proteins 4 % PA stacking gel “loaded” pH 6.8 into wells glycinate<X<Cl- 7.5-15 % 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

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

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 - +

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.

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 1-100 mg/ml depending on circumstances single step and few interfering substances protein concentration extrapolated from standard curve sample not recoverable

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

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.

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)

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

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)