Western Blotting Fundamental Principle, How Western Blots Work

1. Principle

Western blotting usually involves two major processes, namely, SDS-polyacrylamide gel electrophoresis and protein blotting and testing.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Electrophoresis separation describes a phenomenon that charged particles move towards opposite electrode under the influence of electric field. It is used to separate proteins according to their electrophoretic mobility which depends on charge, molecule size and structure of the proteins. Polyacrylamide gel (PAG) is a three-dimensional mesh networks polymer composed of acrylamide and a cross-linker (methylene bisacrylamide) under the catalyzation of ammonium persulfate. PAG is a versatile supporting matrix due to its stable hydrophily and little adsorption and electroosmosis effect provided by its neutrally charged nature. (It possesses several electrophoretically desirable features that make it a versatile medium. It is a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, and can be prepared with a wide range of average pore sizes).

In the presence of SDS, electrophoretic mobility is mainly based on molecule weight instead of on charge and size of the proteins. SDS is an anionic detergent which could break hydrogen bond within and between molecules to unfold proteins and break up secondary and tertiary structures as denaturing agent and hydrotropy agent. Strong reducing agents such as mercaptoethanol and Dithiothreitol (DTT) could disrupt disulfide linkages between cysteine residues. SDS and reducing agents are applied to protein sample to linearize proteins and to impart a negative charge to linearized proteins. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. This new negative charge is significantly greater than the original charge of that protein.

The electrostatic repulsion that is created by binding of SDS causes proteins to unfold into a rod-like shape thereby eliminating differences in shape as a factor for separation in the gel. Minor axis of all rods, the SDS-protein subunit compound are nearly the same, about 1.8nm. And the length of major axis is in proportion to molecular weight of the protein subunit. Thus electrophoretic mobility of the SDS-protein subunit compound is based on molecular weight, eliminating the influence imposed by size and charge.

The sample to be analyzed is mixed with SDS. And the mixed samples are subsquently treated by related solution. Heating the samples to at least 60°C further promotes protein denaturation and depolymerization, helping SDS to bind and enabling the rod-shape formation and negative charge adherence. A bromophenol blue dye may be added to the protein solution to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run. An appropriate amount of glycerol is added to increase density and accelerate the migration of sample solution.

A buffer system with different pH values is applied in gel electrophoresis process. A very widespread discontinuous buffer system is the tris-glycine or "Laemmli" system that stacks at a pH of 6.8 and resolves at a pH of ~8.3-9.0. A drawback of this system is that these pH values may promote disulfide bond formation between cysteine residues in the proteins because the pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g., bis-tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pH values is that the acrylamide gel is more stable at lower pH values, so the gels can be stored for long periods of time before use.

As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the positive electrode (anode) in the lower chamber, the leading ion is Cl¯ ( high mobility and high concentration); glycinate is the trailing ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border between the Cl¯ of the gel buffer and the Gly¯ of the cathode buffer. Because of the voltage drop between the Cl- and Glycine-buffers, proteins are compressed (stacked) into micrometer thin layer-stacking gel layer.

In resolving gel layer, proteins with more negative charges per unit migrate faster than those with less negative charges per unit. That is, proteins with small molecular weight migrate faster than proteins with large molecular weight. The boundary moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel matrix. Stacking and unstacking occurs continuously in the gradient gel, for every protein at a different position.

Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Pore size is controlled by controlling the concentrations of acrylamide and bis-acrylamide powder used in creating a gel. Typically resolving gels are made in 5%, 8%, 10%, 12% or 15%. Stacking gel (5%) is poured on top of the resolving gel and a gel comb (which forms the wells and defines the lanes where proteins, sample buffer and ladders will be placed) is inserted. The percentage chosen depends on the size of the protein that one wishes to identify or probe in the sample. The smaller the known weight, the higher the percentage that should be used. Changes on the buffer system of the gel can help to further resolve proteins of very small sizes.

Western blotting and detection

Five steps are involved in western blotting and detection assay, namely, transfer, blocking, primary antibody incubation, secondary antibody incubation and protein detection and analysis.

Analysis

• Control design:
  Proper control design is essential to western blot. It will guarantee accurate and specific test result by identifying various problems quickly and precisely.
• Control types:
  Positive control: A lysate from a cell line or tissue sample known to express the protein you are detecting. Positive control is designed to verify working efficiency of the antibodies.
• Negative control: A lysate from a cell line or tissue sample known not to express the protein you are detecting. Negative control is to check antibody specificity. Nonspecific binding and false positive result will be identified.
• Secondary antibody control (No primary antibody control): The primary antibody is not added to the membrane. Only secondary antibody is added. This is to check secondary antibody specificity. Nonspecific binding and false positive result caused by secondary antibody will be indicated.
• Blank control: Both primary and secondary antibody are not added to membrane. This is to check membrane nature and blocking effect.
• Loading control: Loading control is used to check sample quality and the performance of secondary antibody system.

Loading controls are antibodies to "house-keeping proteins", or proteins that are expressed at equivalent levels in almost all tissues and cells. Loading controls are required to check that the lanes in your gel have been evenly loaded with sample, especially when a comparison must be made between the expression levels of a protein in different samples. They are also useful to check for even transfer from the gel to the membrane across the whole gel. Where even loading or transfer have not occurred, the loading control bands can be used to quantify the protein amounts in each lane. For publication-quality work, use of a loading control is absolutely essential.

Loading control	Molecular weight (KD)	Sample type
Beta-Actin	43KD	Whole Cell/cytoplasmic
GAPDH	30-40KD	Whole Cell/cytoplasmic
Tubulin	55KD	Whole Cell/cytoplasmic
VCDA1/Porin	31KD	Mitochondrial
COXIV	16KD	Mitochondrial
Lamin B1	16KD	Nuclear (Not suitable for samples where the nuclearenvelope is removed.)
TBP	38KD	Nuclear (Not suitable for samples where DNA is removed)

	Beta-Actin antibody Boster ECL western blot substrate
	GAPDH antibody Boster ECL western blot substrate

3. Detailed western blotting protocol

Preparation of proteins from cell culture

1. Culture cells in tube until the density is up to 80%. Digest cells with 0.05% trypsin (AR1007) or scrape adherent cells off the tube using a cold plastic cell scraper. Wash cells with pre-cooling PBS (AR0030) for two times, centrifugate at 3000rpm for 2-3 minutes, and remove supernatants. Then gently transfer the cell precipitation into a pre-cooled tube. Add mammal cell protein extraction reagent (AR0103) which is 5-7 times the volume of the precipitation into the tube, blow and beat repeatedly.
2. Using ultrasonic processor to break up if the solution is murk. Ultrasonic processes for two seconds, and pauses for two seconds. The power is 100-120 watt. And turn off the power when the murk solution becoms clear. Then lysate the solution on ice for 4-5 hours.
3. Ultrasonic processes the solution again. Centrifugate at 10000rpm for 10 minutes. Discard lipid on the top and lower cell debris, and take out protein solution in the middle. Mix the protein solution with Boster SDS-PAGE loading buffer 2X (AR0103) in the same volume or with SDS-PAGE loading buffer 5X (AR1112) in volume ratio of 4:1 evenly. And then denature the solution in 100? water bath for 5 minutes. (The solution could be used immediately or be aliquotted for store. At -20°C for several months, and at 4°C for 1-2 weeks.

Preparation of proteins from tissue

1. Place surgically resected tissues in pre-cooling normal saline immediately. Wash away blood on the tissue. Chop the tissue into several small pieces (0.1-1g) after weighting. Add Boster enzyme inhibitor cocktail prior to homogenate. And Add 10ml mammal tissue protein extraction reagent (AR0101) per 1g of tissue. Grind the tissue until fully homogenized.
2. Lysate tissue homogenate on ice for 4-5 hours after treated by laultrasonic processor.
3. Centrifugate at 10000rpm for 10 minutes and take out protein solution in the middle. Mix the protein solution with Boster SDS-PAGE loading buffer 2X (AR0103) in the same volume or with SDS-PAGE loading buffer 5X (AR1112) in volume ratio of 4:1 evenly. And then denature the solution in 100 water bath for 5 minutes.

Protein Assay

1. After protein sample collection, concentration of proteins should be quantified to ensure equivalent loading amount of each protein.
2. The most commonly used assay kits are boster BCA protein assay kit (AR0146), micro BCA protein assay kit (AR1110) and Commassie plus protein assay kit (AR0145) respectively.
3. Commassie plus protein assay is ease to carry out and to consume less sample. However, different proteins perform with great variety and poor standard curve linearity will be generated. PH value may change under the function of a variety of elements such as, high concentration of Tris, EDTA, urea, glycerol, sucrose, acetone, ammonium sulfate and detergent. And this will subsequently influence color development and interfere protein quantification.
4. BCA protein assay is valued by its stronger stability, higher sensitivity, higher flexibility. And colored complex formed is more stable with little interfering substance. BCA protein assay has an obvious advantage over commassie plus protein assay, that is, not influenced by detergent.

1. Prepare resolving gel in proportion, blend solution gently to avoid involving too much oxygen. Pour gel solution into two-layer glass pane in gel apparatus slowly. Add a sheet of water to prevent inhalation of oxygen. Placing horizontally, incubate at 37°C for 60 minutes till gel solution is fully polymerized and concreted.

2. Prepare stacking gel in proportion, blend solution gently. Absorb water on concreted resolving gel with filter. Pour stacking gel solution into two-layer glass pane in gel apparatus slowly. Insert comb into the gel carefully (bubble is prohibited between comb tooth. Incubate at 37°C for 60 minutes till gel solution is fully polymerized and concreted and take out the comb gently.

   Note: Boster SDS-PAGE gel preparation kit (AR0138) is recommended for preparation of gel.

Add Boster loading buffer into electrophoresis chamber to a level above sample loading well between two-layer glass pane. Ensure bottom of the gel is immersed in the loading buffer. Liquid level inside the two-layer glass pane is higher than outside level. And then load samples which include treated sample, tissue and cell sample protein for positive control, recombinant protein for positive control and Marker in wells. Load 30-50ug protein to each well and the total loading volume is less than 30ul.

Note: the time should be short as much as possible to prevent diffusion of samples.

After loading samples, place anode and cathode of electrophoresis chamber cap appropriately. And electrophoresis is carried out under proper voltage. In discontinuous system, voltage for stacking gel (70-80V) is lower than voltage for resolving gel (90-110V) to ensure that proteins are concentrated on the same level before running into resolving gel. Run the gel, and when bromophenol blue dye molecule reaches the bottom of the gel, the power is turned off. This process usually takes 2-3 hours.

After electrophoresis, Boster commassie blue staining solution(AR0140), commassie blue fast staining solution(AR0107) and protein silver staining reagents(AR0171) are applied for Gel staining. This is to check if electrophoresis works. Stained gel can not be involved in subsequent membrane transfer procedure.

Wet transfer

1. Take out gel and immerse the gel in Boster transfer buffer (AR1149) for 15-30 minutes.

2. Based on a sandwich model, install electric transfer clip by stacking a sandwich of sponge/transfer pad (filter paper)/gel/NC membrane/transfer pad (filter paper)/sponge which is clamped tightly, no air bubbles within it. Ensure the gel is closest to the cathode and membrane closest to the anode.(Please the following image).

   Note: Sponge, filter paper and NC membrane should be immersed in transfer buffer thoroughly.

3. Add transfer buffer to the top of electrotransfer tank and insert electric transfer clip in it. Place the tank at -20?. Membrane should be closest to the anode. Negative charged amino acid and protein will migrate towards the anode.
4. Constant current of 150-300mA is usually applicable. Transfer time depends on gel concentration.
5. Membrane staning is recommended to check transfer efficiency. Place NC membrane in Boster imprinted membrane fast reversible protein staining reagent (AR0142) for 5-10 minutes and visible red band will appear. The membrane may be distained completely by repeated washing in wash buffer.

1. Rinse blotting membrane: rinse blotting membrane three times using Boster wash buffer at room temperature for 10 minutes.
2. After rinsing, immerse the blotting membrane in Boster blocking buffer. Incubate for 1.5-2 hours at 4°C under agitation.

After being blocked, the membrane is incubated with a primary antibody witch binds to the target protein, and then incubated with enzyme conjugated secondary antibody (HRP conjugated antibody and AP conjugated secondary antibody).

1. Dilute primary antibody in Boster antibody dilution buffer (AR1017). Incubate the primary antibody and the membrane at 4°C overnight.
2. Wash the membrane with wash buffer for three times, about ten minutes each time .
3. Dilute secondary antibody in Boster antibody dilution buffer (AR1017). Incubate the secondary antibody and the membrane at 4°C for two hours.
4. Wash the membrane for 3-6 times, about ten minutes each time.

The choice of western blot antibody detection methods depends on specific substances that are labeled or bond to the secondary antibody. ECL chemiluminiscent detection system and DAB chromogenic detection system are typically used in western blotting.

Boster ECL chemiluminescent system depends on incubation of the western blot with a substrate that will luminesce when exposed to HRP on the secondary antibody. The light is then detected and captured by photographic film.

1. ECL chemiluminiscent solution: Add 1ml water to 50ul color reagent A and 50ul color reagent color reagent B from Boster hypersenstive WB chemiluminescent substrate (concentrated), and mix well. Or add the same amount of color reagent A and color reagent B from and mix well.
2. Cover the blotting membrane with color reagent solution.
3. Exposure: record test result using autoradiography film or CHEMIDOC in darkroom.

4. Using autoradiography film, exposure time for the specific antigen depends on developing effect ranging from seconds to minutes. Using Boster developing and fixing kit(AR0132), wash the film in developing solution. Stop washing when the band appear. And then risen the film in fixative.

   Note: WB Strippping Buffer (AR0153) is recommended to remove primary and secondary antibodies on the membrane if proteins on the membrane needs to be reused.

	Lane1 : MCF7 Whole Cell Lysate Lane2 : Hela Whole Cell Lysate Lane3 : Jurkat Whole Cell Lysate Lane4 : HT1080 Whole Cell Lysate Lane5 : Colo320 Whole Cell Lysate
	Lane1 : MM453 Whole Cell Lysate Lane2 : MM231 Whole Cell Lysate Lane3 : Hela Whole Cell Lysate Lane4 : HT1080 Whole Cell Lysate Lane5 : Colo320 Whole Cell Lysate
	Lane1 : Rat Ovarian tissue Lysate Lane2 : Human placenta tissue Lysate

Boster ECL western blot substrate

Boster DAB chromogenic detection system:

Boster DAB chromogenic reagent: Add 1 ml water, a drop of reagent A, reagent B, and reagent C. Mix well.