Elektroforesis

ELECTROPHORESIS SDS PAGE AND WESTERN BLOTTING

Introduction

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is probably the worlds most widely used biochemical method. Electrophoresis is a separating protein process based on molecule weight using positive and negative electric charge. SDS is a powerful detergent, which has a very hydrophobic end (the lipid like dodecyl part) and highly charged part (the sulfate group). The dodecyl part interacts with hydrophobic amino acids in proteins. Since the 3D structure of most proteins depends on interactions between hydrophobic amino acids in their core, the detergent destroys 3D structures, transforming what were globular proteins into linear molecules now coated with negatively charged SDS groups. After boiling in SDS proteins therefore become elongated with negative charges arrayed down them, so they will move towards a positive electrode. The low molecular weight protein will go downward to positive charge faster than weight ones. There are generally two gels, the main gel and the stacking gel. The stacking gel is of very low acrylamide concentration and is used to form the wells into which the protein is loaded. The low acrylamide concentration also allows most proteins to be concentrated at the dye front, so that dilute protein samples can be compared to concentrated samples on the same gel. The higher the acrylamide concentration, the main gel, the slower the proteins go through the gel. On lower percentage gels proteins go faster, so you should fix on a percentage that puts the proteins you are interested in somewhere in the resolving region of the gel.

Western blotting identifies with specific antibodies proteins that have been separated from one another according to their weight by electrophoresis SDS PAGE. The term "blotting" refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. The blot is a membrane, almost always of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical charge induces the proteins in the gel to move to the membrane where they adhere. The membrane is then a replica of the gel’s protein pattern, and is subsequently stained with an antibody. Western blotting (also called immunoblotting because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. Western blotting can produce qualitative and semiquantitative data about that protein.

Objectives

A. Electrophoresis

The objectives are practicing biomedic student how to do electrophoresis SDS PAGE and identifying proteins existence and proteins weight of the samples

B. Western blotting

The objectives are practicing biomedic student how to do western blotting and determining specific proteins weight of the samples using specific antibody

Method

A. Electrophoresis SDS PAGE

First, we must collect the samples from sources wanted to identify proteins existence and their weight. Then keep those samples and prepare electrophoresis equipments and ingredients for electrophoresis. The main methods of electrophoresis are making electrophoresis gel (acrylamide gel and stacking gel) and running samples in the electrophoresis machine.

1. Making electrophoresis main gel (acrylamide gel)

Ingredients (in different viscosity percentage):

Main Gel		12.5%		10%	15%
No	Materials	1 slap (μL)	2 slap (μL)	1 slap (μL)	1 slap (μL)
1.	Acrylamide 30%	2063	4126	1650	2470
2.	Tris HCl 1.5 M pH 8.8	1250	2500	1250	1250
3.	Sterile aquades	1635	3270	2050	1230
4.	SDS 10%	50	100	50	50
5.	APS 10%	50	100	12.5	12.5
6.	Temed	10	20	10	10

Mix all main gel materials with micropipette in measurement glass, then put it to space between 2 glasses of electrophoresis machine and wait until the mixture become gel

2. Making electrophoresis stacking gel

Ingredients (in different viscosity percentage)

Stacking Gel 3%		12.5%		10%	15%
No	Materials	1 slap (μL)	2 slap (μL)	1 slap (μL)	1 slap (μL)
1.	Acrylamide 30%	257.5	515	412	620
2.	Tris HCl 1 M pH 6.8	312.5	625	625	625
3.	Sterile aquades	662.5	1325	1425	1220
4.	SDS 10%	12.5	25	25	25
5.	APS 10%	3.75	7.5	7.5	7.5
6.	Temed	2.5	5	5	5

After main gel formed, mix all stacking gel materials with micropipette in measurement glass, then put it to space between 2 glasses of electrophoresis machine above main gel and put a comb-like cover to make wells of the gel. Finally, wait until the mixture become gel.

3. Procedure running samples in the electrophoresis machine

-        20μl sample is added with 20μl RSB (reducing sample buffer) and put in eppendorf

-        Heated in boiling water for 5 minutes

-        Fill samples in electrophoresis gel wells. Here we use: well 1 marker, well 2 H. pillory’s pilli, well 3 Shigella dysentery’s pilli, well 4 Shigella dysentery’s OMP, well 5 S. typhi’s pilli (cut 1), well 6 S. typhi’s (cut 2), well 7 S. typhi’s OMP, and well 8 enterocyte protein.

-        Set up electric electrophoresis machine and running samples in 120 V voltage for 90 minutes

-        Lift gel, then stain with commasie brilliant blue R 250 on shaker for 20 – 30 minutes

-        Move gel to destaining solution on shaker for 1 – 2 hours

-        Destain gel overnight on shaker till the gel being clean from commasie brilliant blue R 250

-        Bands of protein are seen in the gel

-        Count molecular weight of the protein seen in the gel

B. Western Blotting

Western blotting is a specific protein weight detection method starting with electrophoresis SDS PAGE without commasie brilliant blue R 250 staining.

Ingredients:

Nitroselulose membrane, filter papers, ponceau 2%, TBS-skim milk 5%, TBS-tween 0.05%, primary antibody, secondary antibody (peroksidase/biotin conjugate), SAHRP, and TMB substrate

1. Procedure transferring and determining the specific protein

-        Get the electrophoresis SDS PAGE gel of protein marker in well 1, H. pylori’s pili in well 3, Shigella’s pili in well 5, and Shigella’s OMP in well 7

-        Make a sandwich like of filter papers, nitroselulose membrane, electrophoresis gel, and filter papers again and put in electric transferring machine

-        Transfer the proteins from electrophoresis gel to nitroselulose membrane using 0.3 A current and 20 V voltage for 2 hours

-        Get the nitroselulose membrane, add ponceau 2% to the membrane to make sure that the proteins are already transferred, and cut the marker

-        Wash with H₂O till the membrane clear from ponceau

-        Add TBS-skim milk 5% as blocking agent of other non protein membrane sides, overnight in 4^o C temperature

-        Wash with TBS-tween 0.05% for  2x10 minutes and shake gently

-        Incubate with primary antibody (49 kDa polyclonal antibody) overnight in 4^o C temperature

-        Wash with TBS-tween 0.05% for  2x10 minutes and shake gently

-        Incubate with secondary antibody for 2 hours and shake gently in room temperature

-        Wash with TBS-tween 0.05% for  2x10 minutes and shake gently

-        Incubate with SAHRP for 1 hour and shake gently in room temperature

-        Wash with TBS-tween 0.05% for  2x10 minutes and shake gently

-        Add TMB substrate for 20 minutes in dark room

-        Stop with aquadest and dry

-        Specific protein bands are seen in the membrane

-        Count specific proteins weight seen in the membrane

Result and Discussion

A. Electrophoresis SDS PAGE

In electrophoresis gel, we can see some bands of protein molecule in different weight. The downer the bands of protein, the lower is molecule weight.

Picture 1 Electrophoresis SDS PAGE result. From left to right, well 1 is protein marker, well 2 is H. pylori’s pili, well 3 is Shigella’s pili, well 4 is Shigella’s OMP, well 5 is first cutting of S. typhi’s pili, well 6 is second cutting of S. typhi’s pili, well 7 is S typhi’s OMP, well 8 is Enterocyte protein

We can cont the protein molecule weight with Linier-regression analysis

-        Protein marker (well 1)

Band	Marker MW	Tracking Distance	Log MW	RF
1	260	2	2.414973348	0.029412
2	135	6.5	2.130333768	0.095588
3	95	8	1.977723605	0.117647
4	72	10.5	1.857332496	0.154412
5	52	17	1.716003344	0.25
6	42	21.5	1.62324929	0.316176
7	34	26	1.531478917	0.382353
8	26	31.5	1.414973348	0.463235

Tracking distance is a distance between border of main gel-stacking gel and protein bands in millimeters. Refractory factor (RF) is tracking distance divided with total distance (from border of main gel-stacking gel to the end of main gel). Then with Microsoft excel program, we can make a graph of the marker result and make a formula to count protein molecule of the samples.

The X axis is log of Molecular Weight (MW) and the Y axis is RF. Now, we find the formula, y = -0.3487x + 1,0303 and R² is 0.91. From that formula, if R² is limit to 1, so more accurate is the formula. The limit accuracy of the formula is R² = 0.97, so we have not accurate of formula, may be because the new biomedic students are not trained yet to add the samples to the electrophoresis gel.

-        H. pillory pilli (well 2), the most dominant protein sample bands

Band	Tracking Distance	Log MW	RF	Protein MW
1	4	2.217297878	0.058823529	164.929324
2	10.5	1.999059898	0.154411765	99.7837676
3	13	1.915122213	0.191176471	82.2474067
4	15.5	1.831184529	0.227941176	67.7929494
5	19	1.71367177	0.279411765	51.7215784
6	30	1.344345958	0.441176471	22.0976432

With the formula, we count the molecule protein of the samples. First, we find the protein samples RF with divide the tracking distance and total distance. Then RF, as X axis, is added to the formula, and we find log MW. Finally, return the log MW with anti-log and now, the protein molecule of the samples is known.

B. Western Blotting

With the same way, we can count the specific protein molecule of the samples because the principle concept is same between western blotting and electrophoresis.

Linier-regression analysis for spesific protein weight counting

Picture 7 Western Blotting result. From right to left, well 1 is protein marker, well 2 is empty, well 3 is H. pylori’s pili, well 4 is empty, well 5 is Shigella’s pili, and well 6 is empty, and well 7 is Shigella’s OMP. Only well 5, Shigella’s pili and well 7, Shigella’s OMP seen in the western blotting result because the 49 kDa polyclonal antibody given to all sample is only bind with specific protein of 49 kDa on Shigella’s protein membrane in OMP and pili.

We can also count the protein molecule weight with Linier-regression analysis

-        Protein marker

Band	Marker MW	Tracking Distance	Log MW	RF
1	260	5	2.414973	0.075758
2	135	8	2.130334	0.121212
3	95	10	1.977724	0.151515
4	72	12.5	1.857332	0.189394
5	52	18	1.716003	0.272727
6	42	22.5	1.623249	0.340909
7	34	27	1.531479	0.409091
8	26	33	1.414973	0.5
9	17	43.5	1.230449	0.659091
10	10	60	1	0.909091

Tracking distance is a distance between border of main gel-stacking gel of electrophoresis gel projected in the membrane and protein bands in millimeters. Refractory factor (RF) is tracking distance divided with total distance (from border of main gel-stacking gel to the end of main gel of electrophoresis gel projected in the membrane). Then, same as electrophoresis, we can make a graph of the marker result and make a formula to count protein molecule of the samples with Microsoft excel program.

The X axis is log of Molecular Weight (MW) and the Y axis is RF. Then, we find the formula, y = -0.5934x + 1,3655 and R² is 0.90. From that formula, if R² is limit to 1, so more accurate is the formula. Same as electrophoresis linier-regression formula, the limit accuracy of the formula is R² = 0.97, so we have not accurate of formula, may be because the new biomedic students are not trained yet to add the samples to the electrophoresis gel.

-        Sample 1, Shigella pili

Band	Tracking Distance	RF	Log MW	Protein MW
1	8	0.121212	2.09745	125.1555
2	12.5	0.189394	1.982472	96.04445
3	13.5	0.204545	1.956922	90.55692
4	14.25	0.215909	1.937759	86.64803
5	34.5	0.522727	1.420359	26.32442
6	38.5	0.583333	1.318156	20.80445

With the formula, we count the specific molecule protein of the samples. First, we find the protein samples RF with divide the tracking distance and total distance. Then RF, as X axis, is added to the formula, and we find log MW. Finally, return the log MW with anti-log and now, the protein molecule of the samples is known. Actually, the 49 kDa antibody only bind with 49 kDa protein of samples. But this antibody is polyclonal antibody, so we can suggest that this 49 kDa polyclonal antibody can react with other epitopes in the pili

-        Sample 2, OMP Shigella

Band	Tracking Distance	RF	Log MW	Protein MW
1	34.5	0.522727	1.420359	26.32442
2	38.5	0.583333	1.318156	20.80445

Same as sample 1, the protein samples RF with divide the tracking distance and total distance. Then RF, as X axis, is added to the formula, and we find log MW. Finally, return the log MW with anti-log and now, the protein molecule of the samples is known. The 49 kDa polyclonal antibody can also react with other epitopes in the shigella’s OMP

Conclution

A. Electrophoresis SDS PAGE

With electrophoresis method, samples of protein in each well is now identified. There are 6 kinds of different protein molecule with different weight in H. pylori’s pili. The proteins weight are 22.1 kDa, 51.7 kDa, 67.8 kDa, 82.3 kDa, 99.8 kDa, and 164.9 kDa.

B. Western Blotting

With western blotting method, specific proteins of 49 kDa antibody is now identified in Shigella’s pili and Shigella’s OMP. 40 kDa polyclonal antibody given to samples suggested react with other different protein molecule weight, 20.8 kDa, 26.3 kDa, 86.6 kDa, 90.6 kDa, 96 kDa, 125 kDa on Shigella’s pili and 20 kDa, 26.3 kDa on Shigella’s OMP.