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Western blot FAQ
420
I、 High background (continuous diffuse bands at non-target bands)
Analysis of high background can be divided into the following reasons.
Antibody causes
1. The use of high antibody concentrations, resulting in non-specific antibody binding
By diluting the antibody, the concentration of the first antibody or the second antibody is decreased, or the appropriate antibody concentration is selected by gradient diluting the antibody.
Membrane and reagent selection problems
1. Improper choice of blocking fluid
Skim milk and animal serum may cross-react with different primary antibodies or contain biotin in blocking solution. Alternative closed reagents such as BSA can be used instead.
2. Improper choice of buffer system
Phosphate buffer interferes with Alkaline phosphatase activity. When using Alkaline phosphatase, reagents containing phosphate should be avoided.
When detecting phosphorylated proteins, avoid using phosphate buffers and blocking agents containing phosphorylated proteins.
3. Membrane fouling results in high background values
By avoiding membrane fouling, clean tweezers are used throughout the process.
Replace a new membrane with enough liquid to avoid membrane drying during operation
In the experiment, mutual interference and friction between the membrane surfaces are avoided
Choose membrane with suitable pore size
Experimental flow problems
1. Inadequate washing of nonspecific sites
Increase the concentration of protein in the blocking solution.
Optimize closing time and temperature. Seal at room temperature for 1-2 hours or 4 ° C overnight.
Inadequate rinsing results in a high background, increasing rinsing times and the volume of rinsing buffer.
Adding detergents to the blocking solution helps reduce background and remove nonspecific binding. Choosing the right concentration of descaling agent can effectively reduce the stripe background
2. The chemiluminescent substrate signal is too strong
Reduce the exposure time of imprinting, reduce the color rendering degree of weak signal.
When the color is too sensitive, you can use a lower sensitivity of the substrate.
Optimize the incubation time between membrane and substrate.
3. Adjust the incubation time of primary and secondary antibodies to avoid too long incubation time.
II、 No signal or faint bands
Antibody causes
1. First antibody and second antibody itself activity problems, such as antibody reagent itself activity is insufficient, antibodies have exceeded their shelf life, antibody repeated use, antibody contamination, antibody use of inappropriate concentration, etc. You can follow the instructions by selecting a quality-assured and activity-proven antibody reagent.
2. The first problem is that the first antibody and the second antibody can not recognize each other. For example, the first antibody can select mouse antibody, the second antibody can select rabbit antibody or recognize other species antibody.
Sample problem
1. The protein of interest doesn't exist.
Through the preliminary investigation, it is confirmed whether the sample contains the target protein.
Protein expression levels in tissues or cell lines are low enough.
2. Insufficient protein in the sample
Ensure that at least 20-30 μg of protein is loaded per lane, using Protease inhibitor to avoid degradation during sample extraction, such as phosphorylated protein detection, protein extraction, phosphatase inhibitors need to be added to avoid sample degradation.
A proper systematic control was set up in each experiment to exclude the influence of other factors.
3. Insufficient lysis of protein extraction samples
Ensuring that the sample is sufficiently cleaved is essential in the process of making sure that the harder-to-extract membrane-bound proteins and organelles (the nucleus and mitochondria) contain less of them.
4. Sample protection time is too long, resulting in sample degradation, or protein deposition, etc. .
Experimental operation
1. The selection conditions are not suitable in the process of film transfer, and the conditions such as film transfer time and voltage can be adjusted
2. When the temperature was too high, the protein sample was degraded.
3. Change the type of membrane , choose the appropriate pore size of the membrane.
4. Chemiluminescent reagents lack sensitivity, or even failure, or color development time is short.
5. The use of detergents in the process of washing concentration is too high, or washing the number of times too long, and so on
6. The buffer system is not compatible with the detection of secondary antibody coupling enzyme. Some buffers contain reagents that may interfere with the test. For example, sodium azide is an inhibitor of HRP and is not suitable for use with HRP-coupled secondary antibodies.
III、 Nonspecific or diffuse bands
Antibody causes
1. Antibody concentration is too high . Using a proven primary antibody. Change the antibody with better specificity.
2. Background signals caused by nonspecific binding of secondary antibodies reduce the concentration of secondary antibodies used, especially the concentration of HRP or AP labeled secondary antibodies.
3. Select cross-validated secondary antibodies. Avoid using primary antibodies produced by closely related, highly homologous species.
Sample reason
1. The excessive amount of gel hole needs to reduce the amount of added samples and reduce the non-specificity of samples.
2. The chemiluminescence substrate signal is too strong to reduce the imprinting imaging or exposure time.
3. There are different isoforms, or variants of the protein, including different molecular weight isoforms, or the protein exists in the form of polymer, the reduction is not sufficient, etc. .
4. Shorten the incubation time between membrane and substrate.
5. The diffuse bands may be caused by high voltage, high migration speed and high electrophoresis temperature.
IV、 Bands appear white.
When the band appears anti-white, it is often the antibody concentration is too high, the substrate is rapidly consumed, the remaining light signal is reduced during detection, resulting in a low middle and high surrounding when the protein is imaged, forming a hollow sample, or a white sample, dilute the antibody to its optimum concentration.
V、 Trailing effect
1. Glycosylated protein
Different glycosylation results in the band-tail effect at the molecular weight higher than predicted.
2. Sample degradation
The presence of Protease inhibitor in the sample helps prevent protein degradation. The long-term storage of lysates leads to the increase of protein degradation products, and the tail effect occurs when the molecular weight of Lysates is lower than the predicted molecular weight.
3. The method of protein sample extraction was optimized to find the suitable way of protein lysis
4. The electrophoretic buffer was reused too many times and consumed too much salt solution.
5. The improper concentration of the separating gel results in the unsatisfactory separation effect of the protein.
VI、 Black dots or speckled background
1. Transfer sponge pollution, when transfer sponge used after not in time gout dry, may grow fungi and other plaque.
2. Buffer contamination, using buffer too often or too long to prevent the growth of microorganisms, so that these spots develop color on the film to form spots.
3. Inadequate dissolution of the blocking solution, such as skim milk, contains granular clumps, resulting in incomplete sealing.
VII、 The band appears as a smiley face
1. The voltage may be too high during migration. The protein migrates too fast because the voltage is not selected according to the molecular weight of the protein.
2. The voltage is too high and the migration rate is too fast, resulting in edge effect. Check the protocol of the recommended voltage and select the appropriate voltage conditions.
3. The gel may be too hot during migration. If the temperature is too high, the pH value of the buffer may change, resulting in the impact of the protein migration environment, the latter can be carried out on ice at 4 ° C electrophoresis, to ensure the environment temperature.
4. If the gel time is not enough, the internal cooling density of the gel is not uniform or the methanol concentration is low, the methanol concentration can be increased appropriately.
VIII、 The strip is not on a horizontal line
1. The migration velocity of the samples in the electrophoresis process is different because of incomplete gel-setting, expired AP reagent, poor gel-setting quality, or incomplete mixing of the gel-setting, impurities in the glass plate, and bubbles.
2. In the process of electrophoresis, the use of excessive voltage, resulting in poor sample separation, temperature and pressure lead to excessive deformation of the gel in the use of the process, should be appropriately lowered the voltage, ice bath environment.
3. When the concentration of salt in the sample is in a suitable range, it is suggested to use a fresh electrophoresis buffer and sample preparation buffer when the concentration of salt is too high and the electrophoretic rate changes or leaks like adjacent lanes.
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