Determination of unknown molecular weight of proteins by SDS page electrophoresis PDF

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Determination of unknown molecular weight of proteins by SDS page electrophoresis. this was done in a practical...

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DETERMINATION OF UNKNOWN MOLECULAR WEIGHT OF PROTEINS BY SDS PAGE ELECTROPHORESIS AND NATIVE GEL ELECTROPHORESIS ABSTRACT Electrophoresis is the process of migration of charged molecules through solutions in an applied electric field. Electrophoresis is often classified according to the presence or absence of a solid supporting medium or matrix through which the charged molecules move in the electrophoretic system.SDS PAGE and Native PAGE were used to anlyse FCS,BSA and albumin and to plot a standard graph of molecular weight against retention factor values.Prepared gels were stained using coomassie blue solution.It was used because of  its

particular properties. Native PAGE gel had more bands compared to SDS page gel.This is because native gel separates proteins according to size and charge whilst for SDS PAGE separation is based on size only INTRODUCTION Electrophoresis is the process of migration of charged molecules through solutions in an applied electric field. Electrophoresis is often classified according to the presence or absence of a solid supporting medium or matrix through which the charged molecules move in the electrophoretic system. The rate of migration of particle depends on the strength of the field, on the net charge size and shape of the molecules and also on the ionic strength, viscosity and temperature of medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species and as a separation technique. It provides the basis for a number of analytical techniques used for separating molecules by size, charge, or binding affinity, example- for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric field applied to a gel matrix. Gel matrix used mainly is polyacrylamide and agarose(Flurkey, W.H., 1990)

"Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS .In native PAGE the mobility depends on both the protein's charge and its hydrodynamic size.If native PAGE is carried out near neutral pH to avoid acid or alkaline denaturation, then it can be used to study conformation, self-association or aggregation, and the binding of other proteins or compounds. Using native gels, it is possible to recover proteins in their native state after the separation (Neuhoff et al.,1988)

A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass. The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field.

Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Because the charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides (Raikos et al., 2006) Samples used for electrophoresis were BSA,FCS and ovalbumin for the experiment.The polyacrylamides created have a wide range of high molecular weights which change its physical and chemical properties.Polyacrylamides are created to suit many industrial applications.Polyacrylamide gel electrophoresis provides very high resolution of DNA molecules 10–3,000 bp long. Under the appropriate conditions, DNA molecules differing in size by only a single base pair can be resolved. Polyacrylamide is used in genetics, genetic engineering and

molecular biology laboratories as a matrix for separating nucleic acid components during DNA sequence analysis and protein identification known as gel electrophoresis.Polyacrylamide is used in genetics, genetic engineering and molecular biology laboratories as a matrix for separating nucleic acid components during DNA sequence analysis and protein identification known as gel electrophoresis.Polyacrylamide is used largely as a flocculator. Flocculators are substances that aid the separation of suspended solids from aqueous systems.Water treatment facilities , as a flocculant in treatment of sewage, waste and drinking water. (This is the greatest use of acrylamide in water purification to flocculate suspended organic matter.) They can be used for mill sizing of paper, paper coating, paperboard, paper and board manufacture for packaging materials.Ore processing, oil recovery, crude oil production processes(Raikos et al., 2006)

MATERIALS AND METHODS Preparation of samples Table 1.A table showing volumes for the preparation of samples Reagents

Separating Gel

Native Gels: Acrylamide monomer solution: Separating gel buffer: Distilled water:

2.5 ml 1.9 ml 3 ml

ONLY for SDS Gels: As above but add 10% SDS before TEMED and APS

75 μl

To be added last: 5 μl TEMED APS

50 μ

TEMED and APS were added to the separating gel solution and the flasks were swirled gently to mix in the catalysts evenly without aerating the solution.The solution was then

pipetted carefully in between the two glass plates of the cassette with a Pasteur pipette ensuring that no air bubbles are present. Immediately 3 were then pipetted.Drops were then put on on top of gel layer to ensure a straight surface.The gel was then allowed to polymerise for 20 minutes. After polymerisation the butanol phase was then washed from the surface several times with water bottle (dH2O). The stacking gel solution was then prepared as in table following the same procedure as for separating gel. Table 2.A table showing volumes needed for preparation of stacking gel Reagents

Stacking Gel

Native Gels Acrylamide monomer solution Stacking gel buffer Distilled water

0.4 ml 0.63 ml 1.5 m

ONLY for SDS Gels: As above but add 10% SDS

To be added last: TEMED APS

25 μl

5 μl 20 μl

The stacking gel solution on top of the polymerised separating gel was then pipetted with a clean Pasteur pipette. Immediately the teflon “comb for wells” was inserted into the stacking gel between the 2 glass plates. The gel was allowed to polymerise for 15 min. After polymerisation the comb was carefully removed. The protein samples were prepared as follows into microcentrifuge tubes Table 3.A table showing volumes needed for preparation of protein samples NATIVE- PAGE

Sample (µl)

Sample Buffer (µl) Dye (µl)

Sample Buffer (µl) Dye (µl)

FCS

5

75

10

BSA

20

20

5

Ovalbumin

75

5

10

Mixture

FCS: 5 BSA: 20 Ovalbumin: 75

100

25

Table 4 .A table showing calculated values for protein samples with dyes SDS-PAGE

sample (µl)

Buffer (µl)

Dye (µl)

SDS (µl)

B-mercaptoe thanol (µl)

FCS

5

75

10

32

8

BSA

20

20

5

16

4

Ovalbumin

75

5

10

32

8

Mixture

FCS : 5 BSA: 20 Ovalbumin: 75

100

25

80

20

To each sample glycerol was added.SDS samples were boiled for 3 min and then cooled. Native samples were kept on ice 15 minutes. Sample loading and electrophoresis The electrophoresis system was assembled and the tank buffer was poured into the reservoirs. Buffer was allowed to flow over the sample wells. Samples were then applied to individual wells with an automatic pipette (20 µl per sample).Electrodes were then connected .The tracking dye (bromophenol blue) was then followed and power was switched off when the front reached + 0.5 cm. Gel was transferred into plastic container and 50 ml of Fairbanks A solution was then added.. I t was then microwaved on high for 2 minutes, or until solution reached boiling point.It was then removed from microwave and allowed b to coolfor 5 minutes at room temperature, with gently shaking. The solution was then discarded and the gel was rinsed in distilled water The steps were repeated with dH2O. 24. 100ml of Fairbanks B solution was added and steps 3 and 4 were again repeated. Finally Fairbanks D solution (destaining solution) was added and steps 3 and 4 were repeated until clear background is obtained.

RESULTS

Fig 1 A diagrammatic representation of SDS page electrophoresis gel

Fig 2 A diagrammatic representation of Native page electrophoresis gel

Table 5 A table showing number of bands for both SDS and Native gel page and their calculated Rf values. Lane no

Content

Number of bands

Migration front Rf = (mm) distance front

migration migration

Native PAGE 1

BSA

3

2

FCS

2

3

Ovalbumin

4

4

Mixture

6

Lane no

Content

Migration distances(mm)

Migration front Rf = (mm) distance front

migration migration

1

BSA

25

90

0.28

2

FCS

25

90

0.28

30

90

0.33

55

90

0.61

21

90

0.23

40

90

0.44

79

90

0.88

21

90

0.23

25

90

0.28

30

90

0.33

39

90

0.43

55

90

0.61

78

90

0.87

SDS PAGE

3

4

Ovalbumin

Mixture

Table 6. Table of Rf values and migration distances with molecular weights for the known markers sample. Band

Migration

Migration

R=

no

Distance

front (mm)

distance migration weight ( kDa )

(mm)

migration Molecular

Log Mr

front

1

7

85

0.08

250

2.40

2

17

85

0.2

150

2.18

3

26

85

0.31

100

2.00

4

23

85

0.27

70

1.85

5

43

85

0.51

50

1.70

6

50

85

0.59

40

1.60

7

60

85

0.71

30

1.48

8

64

85

0.75

20

1.30

9

71

85

0.84

15

1.18

10

78

85

0.92

10

1.00

Table 7. Table  showing the size of the proteins in Daltons from the standard curve determined from the SDS PAGE Lane no.

Content

Number

of Mr (kDa)

bands 1

MW

10

250 150 100 75 50 37 25 20 15 10

2

BSA

1

112

3

FCS

3

112 93.3 32.4

4

Ovalbumin

3

135 63 12

5

Mixture

6

135 112 93.3 61 12.3

DISCUSSION Ovalbumin is a monomer, globular phosphoglycoprotein with molecular weight of 44.5 kDa.Bovine albumin serum is 66.4kDa and molecular weight for fetal calf serum is 68.7kDa according to ().Native PAGE gel had more bands compared to SDS page gel.This is because native gel separates proteins according to size and charge whilst for SDS PAGE separation is based on size only.This is because Native gels are run in non-denaturing conditions, so that the analyte natural structure is maintained. This allows the physical size of the folded or assembled complex to affect the mobility while for SDS separation is only based on size since all proteins will be denatured hence all will have negative charge.(Shagger.,H,1995).

In FCS there were three bands indicating that there are three types proteins in it while in BSA there was one band indicating that there is one protein in BSA and 3 types in ovalbumin since there were 3 bands.The mixture contained 4 bands indicating that there were three types of proteins inside the mixture.Proteins in BSA had almost the same weight since from the SDS gel there was only one band.This is inline with(Raikos et al,2006) who suggest that separation of SDS is based on size only.BSA moved a larger distance because it has a lighter mass compared to FCS and BSA. Coomassie blue  creates a rapid and convenient staining procedure. This capability of G-250  is due to its particular properties. Coomassie  G-250 manifests a leuco form below pH 2. Solutions of the dye, dark blue black at pH 7, turn a clear tan upon acidification. The leuco form recovers its blue color upon binding to protein, apparently due to the more neutral pH of the environment around the protein molecule. Under proper conditions, a gel placed in an acidified solution of Coomassie  G-250 will manifest blue protein bands on a light amber background.(Whitaker, J.R,1963 ). The peroxidsa=The appearance of dark brown bands was caused by peroxidase activity and indicated the presence of a peroxidase isoenzyme in the gel.

Silver staining is another staining procedure which utilises the protein binding properties of ions which are then reduced to silver metal using a developing solution creating a visible image .The primary benefit of silver staining is its high sensitivity,as it is able to detect 1ng of protein (Weiss et al. 2009),making it extremely useful for applications involving low protein levels.However, silver staining involves multiple steps and reagents, making the process relatively time consuming and laborious. In addition, the gel requires developing after staining, in order to visualise the proteins, and the length of time required for developing is highly variable between gels, meaning reproducibility is low.

Band resolution could be improved by doubling the salt concentration in stacking and separating gels, but the gel must be run at lower voltages.During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution. A solution of acrylamide and bis acrylamide is polymerized. Acrylamide alone forms linear polymers. The bisacrylamide introduces crosslinks between polyacrylamide chains. The 'pore size' is determined by the ratio of acrylamide to bisacrylamide, and by the concentration of acrylamide. A high ratio of bis acrylamide to acrylamide and a high acrylamide concentration cause low electrophoretic mobility. Polymerization of acrylamide and bisacrylamide monomers is induced by ammonium persulfate (APS), which spontaneously decomposes to form free radicals. TEMED, a free radical stabilizer, is generally included to promote polymerization.(Weiss et al. 2009) Errors during pipetting could have resulted in errors during the experiment,however taking extra caution using pipettes by using new pipette tip each and every time could minimise errors of the experiment.

CONCLUSION Native PAGE gel had more bands compared to SDS page gel.This is because native gel separates proteins according to size and charge whilst for SDS PAGE separation is based on size only.From the plotted graph of molecular weight and retention factor values ,protein sizes can be found from the graph.Those proteins with high molecular weight travelled short distances whilst those with low molecular weight travelled long distances.Silver

staining is another staining procedure which utilises the protein binding properties of ions which are then reduced to silver metal using a developing solution creating a visible image .The primary benefit of silver staining is its high sensitivity REFERENCES

Flurkey, W.H., 1990. Electrophoretic and molecular weight anomalies associated with broad bean polyphenoloxidase in SDS-PAGE electrophoresis. Phytochemistry , 29( 2), pp.387-391. Neuhoff, V., Arold, N., Taube, D. and Ehrhardt, W., 1988. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis , 9( 6), pp.255-262. Raikos, V., Hansen, R., Campbell, L. and Euston, S.R., 2006. Separation and identification of hen egg protein isoforms using SDS–PAGE and 2D gel electrophoresis with MALDI-TOF mass spectrometry. Food chemistry, 99 ( 4), pp.702-710. Schägger, H., (1995) Native electrophoresis for isolation of mitochondrial oxidative phosphorylation protein complexes. Methods in enzymology , 260, pp.190-202.\ Whitaker, J.R., 1963. Determination of Molecular Weights of Proteins by Gel Filtration of Sephadex. Analytical Chemistry , 35( 12), pp.1950-1953.

Weiss, I., Braun, H.P. and Schägger, H.,( 2009). Blue native PAGE. Nature Protocols , 1 , pp.418-428. Yang, T.H., 2008. Recent applications of polyacrylamide as biomaterials.Recent Patents on Materials Science , 1( 1), pp.29-40. CALCULATIONS RELATED TO THE EXPERIMENT

Calculation: 2.5 ml of the monomer solution Take 29.19 g and 0.81 g of acrylamide and dissolve in some distilled water , top up to 2.5 ml using dH2O 55 ml water saturated butanol Take 50 ml butanol plus 5 ml of dH2O 10% ammonium persulphate 10 g in 100 ml , therefore Dissolve 10 g in some dH2O and make up with dH2O 300 ml N,N,N´,N´-tetramethylenediamine No information provided on how to make it, i think it comes already as a solution 75l of a 10% SDS solution 10 g in 100 ml distilled water , therefore dissolve 7.5mg in some distilled water and make up 75l to using some dH2O 75l of a 2 x Sample buffer for FCS composed of 0.125M Tris-HCl, pH 6.8 containing 10% Glycerol and 0.2% bromophenol blue solution) For Tris : m

Glycerol : 10 %

=

Mr x C x V

=

(121.14 g/mol) x (0.125 M) x (0.000075L)

=

1.14 mg Tris

=

10 ml in 100 ml dH2O, therefore in 75l ml there is 7.5

=

0.2 g in 100 ml dH2O, therefore in 60 ml there is

ml glycerol Bromophenol 0.2% 0.15mg bromo

Weigh out the 1.14 mg Tris and 0.15mg bromophenol blue dissolve it in some distilled water, add 7.5

ml glycerol and top up to 75l with dH2O.

20 µl of 0.1 mg/ml BSA 0.1 mg in 1ml dH2O, therefore dissolve 2 mg and make up to 20 µl with dH2O 5l of 0.1 ml /ml FCS 0.1 ml in 1ml dH2O, therefore add 0.5 l and make up to 5l with dH2O 10ml SDS- Electrode buffer/tank buffer from the original powders 10 g in 100 ml dH2O , therefore For 10 ml , dissolve 1 g in some dH2O and make up to 10 ml using dH2O 500 ml of a 10X stock solution consisting of 25 mM TRIS, 192 mM Glycine (pH 8.1-8.3) ...