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Western Blot / WB protocol

Far Western Blot

Enhanced chemiluminescence (ECL) is a method which provides highly precise detection of proteins from Western blots.

In many luminescenct assays, the light emitted is of low intensity and short duration. We use an enhanced version of the chemiluminescence reaction. This involves performing the reaction with luminol in the presence of modified phenols. The phenols greatly enhance light emission, altering the kinetics of the reaction in such a way that the light emission is much prolonged and very intense.

ECL Western Blot

Enhanced chemiluminescence (ECL) is a method which provides highly precise detection of proteins from Western blots.

In many luminescenct assays, the light emitted is of low intensity and short duration. We use an enhanced version of the chemiluminescence reaction. This involves performing the reaction with luminol in the presence of modified phenols. The phenols greatly enhance light emission, altering the kinetics of the reaction in such a way that the light emission is much prolonged and very intense.

HRP Western Blot


Enzymatic labels are most commonly used for Western blotting and, although they require extra steps, can be extremely sensitive when optimized with an appropriate substrate. Horseradish peroxidase (HRP) is the enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates are available for use with the enzyme. Horseradish peroxidase (HRP) conjugated antibodies are considered superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody due the smaller size of HRP enzyme and compatibility with conjugation reactions. In addition, the high activity rate, good stability, low cost and wide availability of substrates make HRP the enzyme of choice for most applications.

Wet Western Blot

Wet Western Blot
Western blot transfer can be done in wet or semi-dry conditions. In wet western blot transfer, from the image above, you can see the wet western blot transfer modle-a sandwich with a regular order. That's a sandwich of sponge/paper/gel/membrane/paper/sponge, which is clamped tightly, no air bubbles within it. It's important that the gel is closest to the negative electrode and membrane closest to the positive electrode. Semi-dry western blot transfer is generally faster but wet western blot transfer has a less tendency to failure and is especially recommended for large proteins more than 100 kD.

Semi-dry Western Blot

In semi-dry western blot the electrodes are placed directly in contact with the gel/nitrocellulose membrane sandwich to provide a fast, efficient transfer. The polyacrylamide gels must be equilibrated in transfer buffer, to remove electrophoresis buffer salts and detergents, and the nitrocellulose membranes and filter papers arepre-wetted, but that is all the buffer that is required. Using a platinum-coated titanium plate as the anode and a stainless-steel plate as the cathode, the Trans-Blot SD cell transfers in a horizontal configuration without a buffer tank or gel cassettes. Because of this direct contact there is a minimum of transfer buffer required (less than 200ml). It is important to exclude excess moisture and air bubbles trapped in the filter papers and membrane when setting up the transfer, usually a pipet rolled over the surface will take care of this, but other than that, the set-up for this process is extremely simple. Up to four mini gles can be transferred at the same time by placing them sidy-by-side on the anode platform.

Densitometry Western Blot

Western Blot scans need to be converted into grey scale and saved as a JPEG file.

1) Open Western scan in Image.

2) Click the 'rectangular selection' (under file menu) and select a box around a band of interest. Avoid any background if at all possible. The same box size must be able to fit all of the bands you would like to measure.

3) Once you have boxed an area go to the 'analyze' drop menu. Select 'measure'. A results box should pop up.

4) Use the arrow curser to move your box along to the next band of interest, and repeat the 'measure'.

5) Once you have finished measuring all protein bands, move the box onto the background. Measure the area of the background blot (in the same manner). If the background is somewhat blotchy and uneven in colour then select a background area equivalent to that around the protein band.

6) Once all the measurements are finished, select 'all' (control A) and 'copy' (control C) and paste in excel.

7) In excel erase all the values except the Mean values. Subtract all the Mean values from 255 (including the background). Then subtract the background from each band value. This will give you the band intensity;

Fluorescent Western Blot

Fluorescent reagents are growing in popularity for western blotting because they offer increased time savings over chemiluminescence western blot detection and reduced chemical waste compared to both chemiluminescent or chromogenic detection systems.

Fluorescent western blot applications differ from other detection systems in that the signal is not a product of an enzyme reaction, but rather a transient light emission resulting from the excitation and subsequent release of photons as the excited molecule returns back to its normal state. This is in contrast to enzyme-substrate systems that produce a colored product or light emission as a result of an chemical reaction. This allows optimized fluorescent applications to be more quantitative than enzyme systems.

Chemiluminescence Western Blot

Chemiluminescence Western blots are probed with a primary antibody against the target protein, followed by a secondary antibody labeled with HRP (horseradish peroxidase) enzyme. A chemiluminescence substrate for the HRP enzyme is carefully applied to the blot, and light is emitted when the HRP enzyme modifies the substrate. Photographic film or an imaging system using a digital CCD camera captures the emitted light as an image. Most importantly, chemiluminescence yields the greatest sensitivity of any available detection method.

Membrane Protein Western Blot

Membrane proteins, owing to their highly hydrophobic nature, have always posed a daunting challenge to biochemists and structural biologists working on the characterization of these "naughty" proteins.

Western Blot Buffer / Reagents

In western blot, except lysis buffer which is needed in sample preparation, other reagents also have to be prepared for western blot. Loading buffer, running buffer, coomassie brilliant blue staining solution, and coomassie destaining solutionare needed to be prepared for SDS-PAGE, while western blot transfer buffer (recipe here is for wet transfer) preparation is required for protein transfer. Sometimes, ponceau red staining is an alternative to check whether the protein transfer is successful, so a recipe of ponceau red staining solution is necessary. Besides, TBS buffer, blocking buffer, and TBST buffer (washing buffer) are also needed to be prepared. The table below is a recipe especially about buffer or reagent needed in western blot, or we can name this table after "western blot buffer recipe".

Western Blot Sample Preparation

Sample preparation for western blot Western blot sample preparation occurs before SDS-PAGE; it is the start of western blot. The sample exctract can be prepared from cell culture or tissues by mechanical crushing like homogenization and sonication methods or high pressure disruption, and then they are further lysed with some lysis buffer to make sure the maximum percent of proteins have been extracted. Extraction is always performed on ice to prevent protein degradation. Protease and phosphatase inhibitors are added to protect the proteins from digestion by protease that leaked out during sample preparation.

Western Blot Gel Electrophoresis

Western Blot Gel Electrophoresis - SDS-PAGE

SDS-PAGE for western blotSDS-PAGE is a standard means for separating proteins according to their molecular weight. This technique includes one dimensional electrophoresis and two dimensional electrophoresis. One dimensional gel electrophoresis is used for separation of most routine proteins, and here we mainly describe this technique.

Western Blot Transfer

Just as proteins have been bound with electrical charge provided by SDS, they can be transferred in an electrical field from the gel onto a sturdy support, a membrane that "blots" the proteins from the gel. This is called western blot transfer. Here we will talk about methods of western blot transfer, transfer membranes, factors for tansfer efficiency and common western blot transfer systems.

Western Blot Blocking

Membrane blocking in western blot is for the purpose of preventing the non-specific binding of antibodies including both primary antibody and secondary antibody) to the membrane, so that the common problem of high background in western blot can be avoided.

Western Blot Antibody Incubation

Primary antibody binds with antigen after primary antibody incubationAfter being blocked, the western blot membrane is subsequently incubated with a primary antibody - recognition of the target protein.

Western Blot Detection

Western blot antibody detection procedures vary widely. One common variation involves in direct detection and indirect detection. Through the direct detection method, the primary antibody used to detect an antigen on the blot is labeled with fluorescent dye or an enzyme. Most researchers prefer the indirect detection method, the direct detection method is not used widely. In the indirect detection method, to bind to the antigen, a primary antibody is added first, either monochlonal antibody or polyclonal antibody. This is followed by a labeled secondary antibody that is directed against the primary antibody. Labels include biotin, fluorescent probes, such as fluorescein or rhodamine, and enzyme conjugates such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). The indirect detection method has many advantages over the direct detection method.

Antibody
Western blotting / WB Antibody
- Western blotting / WB Antibody Features
- WB Products Center
- Wstern Blot / WB Technique Center
-- Western blot introduction
What is Western Blot
Western Blot for Diagnosis
Western Blot Applications for Research
-- Western Blot / WB tips
Sinobiological Western blot general tips
Western Blotting: 10 Technical Tips for Success
-- Western Blot / WB FAQ
1. Why are there multiple bands on my blot?
2. Why is there a weak signal or no signal at all?
3. Why is there high background and/or diminished signal with anti-phosphor tyrosine antibodies?
4. Why does my antibody activity diminish in Western Blot over time?
5. How do I overcome inadequate or poor transfer problems?
6. How do I avoid "splotchy" or uneven Western Blots?
7. What can I use as positive control lysates for sinobiological WB antibodies?
8. Too many bands on a Western blot
9. No Signal or Weak Signal
10. Nonspecific Bands
11. High Background
12. How much protein should I analyse?
13. What percentage acrylamide gel should I use to resolve the proteins?
14. What membrane should I use?
15. What blocking buffer should I use?
16. What dilution of primary antibody should I use?
-- Western Blot / WB protocol
Far Western Blot
ECL Western Blot
HRP Western Blot
Wet Western Blot
Semi-dry Western Blot
Densitometry Western Blot
Fluorescent Western Blot
Chemiluminescence Western Blot
Membrane Protein Western Blot
Western Blot Buffer / Reagents
Western Blot Sample Preparation
Western Blot Gel Electrophoresis
Western Blot Transfer
Western Blot Blocking
Western Blot Antibody Incubation
Western Blot Detection
-- Western Blot / WB trouble shooting
High Background
Speckled Background
Multiple Bands
No Bands
Low Signal
Other Problems
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"