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What is the difference between monoclonal antibodies and polyclonal antibodies?
Monoclonal antibodies recognize only one specific epitope and have low cross-reactivity. The preparation of monoclonal antibodies is relatively difficult and takes a relatively long time. Polyclonal antibodies recognize multiple epitopes, and have high sensitivity and cross-reactivity. The preparation of polyclonal antibodies is relatively easy and quick.
How to choose between monoclonal antibodies and polyclonal antibodies?
Choose an appropriate antibody type according to the demand of different experiments. Because of their ability to recognize multiple epitopes, polyclonal antibodies can amplify the signal from a target protein with low expression level in samples and facilitate enrichment of target proteins in IP/ChIP experiments. Monoclonal antibodies recognize a specific epitope so that they are more suitable for accurate localization of target proteins within cells.
How to choose a lysis buffer?
For WB sample preparation, epitopes on the antigen should be denatured, so lysis buffers with strong lysis ability are needed, such as RIPA lysis buffer. For IP/ChIP experiments that require natural antigen status, antigens should not be denatured, so mild lysis buffers are needed.
How to prepare a lysis buffer for WB?
First, a lysis buffer should have a pH value similar to physiological pH, and generally the Tris-HCl (pH 7.6) buffer is used. Second, salt ion concentration in lysis buffer should not be too high, and generally, the concentration of physiological saline (150mM, NaCl) is chosen. A certain volume fraction of surfactants should be added to increase protein solubility. Commonly used detergents include SDS, TritonX-100, NP-40, NaDOC, etc. A proper amount of protease inhibitors should be added to inhibit protein degradation.
What is the difference between the commonly used detergents?
The commonly used detergents can be divided into two categories: ionic detergents and nonionic detergents. Ionic detergents such as SDS and NaDOC have strong lysis ability, can break the cell membrane and nuclear membrane, and even denature proteins. Nonionic detergents such as MP-40, TritonX-100 and Tween-20 are relatively weak, but can break membranes. Choose a suitable detergent according to the experimental requirements.
What are the methods for protein quantification?
There are many protein quantification methods, such as Lowry, Bradford, BCA, A280, etc.. According to sample preparation for WB, BCA is less affected by detergents, has a strong anti- interference ability, and is suitable for concentration determination of lysis buffer.
How much sample should be loaded?
For purified protein, it generally needs 100ng. For cell and tissue lysate, it generally needs 10-30μg. The amount of sample loaded should be adjusted according to the actual situation.
How to choose an appropriate SDS-PAGE gel concentration?
SDS-PAGE gel concentration is related to protein molecular weight. Generally, small molecule proteins (100kD) need 8%-6% gel.
How to choose a transfer membrane?
NC membrane and PVDF membrane are commonly used transfer membranes. PVDF membrane has a higher protein binding capacity and a higher sensitivity, and needs to be activated by alcohol solvents. NC membrane does not require activation. PVDF membrane binds proteins mainly through charge interaction, while NC membrane binds proteins mainly through hydrophobic interaction. The former is stronger.
What is the purpose of PVDF membrane activation?
To activate positively charged groups on the surface of PVDF membrane to improve the binding of the membrane to proteins.
Dry transfer or wet transfer?
For proteins with ordinary molecular size, dry transfer or wet transfer have almost equal effects, while dry transfer is more efficient and requires fewer reagents. For proteins with large molecular size, wet transfer works better.
How to choose a suitable blocking buffer for WB?
Commonly used blocking buffers include protein-containing blocking agents, such as BSA, casein and non-fat milk, and protein-free blocking agents, such as Tween-20 and gelatin. Generally speaking, non-fat milk has a relatively complex composition and offers a better blocking effect. BSA has a simple composition and is suitable for most cases. Casein has a poor solubility and needs to be mixed evenly. Under neutral and alkaline conditions, casein is negatively charged and offers a good blocking effect. Furthermore, when choosing a blocking buffer, the characteristics of target proteins and antibodies should also be taken into considerations.
How to increase transfer success rate of small molecule proteins?
Use a transfer membrane with small pore size (0.22μm), and shorten the transfer time.
How to increase transfer success rate of large molecule proteins?
Use low concentration gel, choose wet transfer, reduce the methanol concentration in transfer buffer, add 0.1% SDS to promote protein migration, and extend the transfer time (at 4 ℃ overnight).
How to store protein samples?
Stored at -80 ℃ after denaturation.
How to prepare tissue samples for WB?
Grind the tissue of interest with liquid nitrogen or on ice, add lysis buffer and protease inhibitors to tissue samples, and centrifuge at a high speed and low temperature to obtain tissue lysate.
How to choose an internal control for WB?
There are many types of internal controls. Choose a different internal control for proteins with different localizations. The commonly used internal controls, beta-actin, GAPDH and Tubulin, are used for the cytoplasmic proteins; Lamin B1, Histone and TBP are used for nuclear proteins.
What happens if there are white bands or even yellow bands after imaging and how to solve it?
The amount of protein loaded or primary antibody is too large, leading to immediate consumption of luminescent substrates, resulting in white bands. It is possible to get a better result by adjusting the amount of protein loaded and antibody.
What happens if there are black dots on the background after imaging and how to solve it?
Blocking buffer must be mixed evenly before use. The presence of insoluble particles results in black spots on the background.
What causes high background?
High background may be caused by many factors:① inappropriate blocking buffer or inadequate blocking period;② secondary antibody has non-specific binding, leading to high background;③ antibody incubation period is too long or incubation temperature is too high.④ washing after antibody incubation is not clean, leading to non-specific background;⑤ the membrane is too dry during operation;⑥ large amounts of luminescent substrates remain after luminescent substrate incubation;⑦ the exposure time during imaging is too long;⑧ touch the membrane with bare hand or squeeze the transfer membrane during operation.
What causes no or weak bands?
The possible reasons are as follows:① The content of the target protein is too low in the sample. It is necessary to confirm whether the target protein is expressed in the selected sample and whether the drug induced model is successfully constructed.② Protein transfer efficiency is not high. In particular, for large molecule proteins, it is needed to examine transfer efficiency and optimize transfer conditions.③ The primary antibody incubation time is too short. Long incubation at low temperature is recommended in order to ensure adequate interaction between antigen and antibody.④ Antibody inactivation due to improper storage.⑤ WB detection system is polluted by NaN3, affecting the enzymatic activity of HRP.⑥ The developing solution has been used for a long period. It is important to prepare the solution in time.
Why there are uneven white spots on the blot?
Air bubbles are produced during operation, such as during transfer and during luminescent substrate incubation.
What causes band distortion phenomenon?
The possible reasons include: gel solution preparation is not accurate and gel solidification is too fast, leading to gel concentration inhomogeneity; the interface between concentration gel and separation gel is not smooth; protein solubility in the sample is not good, and it is needed to optimize the extraction conditions; during gel electrophoresis, the voltage is too high, leading to high temperature and rapid band migration.
What causes band trailing phenomenon?
The possible reasons include: there are insoluble substances in protein samples, and it requires centrifugation or optimized sample extraction conditions; too much sample is loaded that extends the volume of each hole; electrophoresis solution has remained for a long period that effective components in it are lost.
Why is the molecular weight of the target protein not consistent with theory?
The possible reasons include:① Experimental system influence. There is an error associated with the Marker used. When gel solution preparation is inaccurate, the separation effect of the gel changes and the indication of the Marker becomes inaccurate.② Protein property influence. The proteins have post-translational modifications like phosphorylation and glycosylation, or have post-translational splicing. The proteins form dimers or polymers. During sample preparation, the formation of dimers or polymers can be eliminated by the addition of β-mercaptoethanol (BME) or DTT. The proteins are charged, and too many aspartic acid and lysine residues may affect the binding of SDS to the proteins, causing that protein migration speed and protein size are not a linear relationship.
How to choose between paraffin sections and frozen sections?
Frozen sections can well preserve antigen activity, and there is no need for antigen repair in the experiment. However, frozen sections require higher storage condition (-80 ℃), are thicker than paraffin sections, and offer worse results than paraffin sections. Paraffin sections can maintain the morphology and structure of tissue cells, but because of the inactivation of antigen by aldehydes and alcohols during sample preparation, paraffin sections need repair treatment. Paraffin sections can be stored at normal temperature and are convenient for use.
Why does it need to dewax and hydrate paraffin sections?
To expose the tissue wrapped by wax and restore the hydration state of tissue cells.
Why should antigen repair be performed?
During normal production of paraffin sections, aldehydes such as formalin are used to fix tissue, leading to the formation of cross-linking between aldehyde reagents and proteins in tissue cells. The cross-linking networks hide antigen epitopes, blocking their binding with antibodies. Therefore, antigen repair is needed to expose antigen epitopes for detection.
How to choose an antigen repair method?
There are two major methods for antigen repair: ① Enzyme repair. A method that cuts off peptides that hide epitopes to achieve antigen repair by using enzymes like trypsin and pepsin. ② Heat repair. A method that reverses the cross-linking between aldehyde reagents and proteins and achieves the reconstruction of secondary structure and tertiary structure of antigen epitopes. Two examples of common methods are microwave treatment and high pressure heating. The former has a good effect on the repair of intracellular antigens or intercellular matrix or basement membrane antigens. The latter is effective for the repair of most antigens, especially nuclear proteins.
Why should endogenous enzymes be removed and how to remove them?
The presence of endogenous enzymes and biotins in the body will interfere with the entire detection system. Therefore, the effect of endogenous enzymes must be eliminated to avoid false positive results. Endogenous peroxidases are common in the liver, spleen, kidneys and other tissues, and their impact can be eliminated by 3% hydrogen peroxide. Endogenous biotins are common in the liver, spleen, kidneys, brain, skin and other tissues, and their interference can be eliminated by avidin soaking before dyeing. Levamisole and tartaric acid can respectively remove the alkaline and acid phosphatase of endogenous phospholipases. In general, 3% of hydrogen peroxide is sufficient to remove the interference of most enzymes.
Which blocking reagent is generally used in IHC?
For IHC experiments, it is recommended to use the serum (consistent with the source species of secondary antibody) as a blocking reagent. Compared with conventional blocking reagents such as BSA and casein, serum not only blocks nonspecific binding proteins but also blocks FC receptors in samples.
Can non-fat milk be used for blocking in IHC?
No, non-fat milk itself contains biotins, which interfere with the detection.
What causes higher staining background and how to solve it?
The possible reasons include: ① Inadequate blocking or inappropriate blocking reagent. It is necessary to optimize blocking conditions. ② Existence of endogenous peroxidase or biotin interference. It is necessary to inactivate endogenous enzymes or prolong treatment time. ③ Antibody incubation concentration is too high or time is too long. It is necessary to optimize the antibody incubation time and concentration. ④ Washing is insufficient, resulting in antibody residue. ⑤ DAB staining time is too long. ⑥ Tissue section is too dry that increases background.
Why is staining effect weak or even negative?
The possible reasons include: ① Low or no expression of the protein of interest in samples. It is needed to confirm the induction effect or amplify the signal; ② Antigen repair is not successful. It is needed to optimize the repair time, temperature and buffer system; ③ Antibody incubation time is too short or antibody concentration is too low. It is needed to optimize antibody incubation time and concentration.
How to control dewaxing?
Generally, xylene or dewaxing agents are used for tissue dewaxing. The dewaxing conditions need to be adjusted according to the season and room temperature. The temperature in summer is higher, so the dewaxing time can be shortened appropriately. Conversely, the dewaxing time can be prolonged in winter. In addition, dewaxing agents need a prolonged time.
How to solve the problem of tissue stripping?
The easiest way is to use anti-stripping slides or anti-stripping reagents, such as APES-acetone solution (1:50). In addition, after high pressure repair, samples should be left to cool naturally, and cannot be washed by cold water because it can easily lead to stripping. Try to operate as carefully as possible during washing.
Why do I need to fix the cell in the experiment?
To keep the protein’s original structure and intracellular location. But it is not necessary for membrane protein.
What are the common fixatives?
The common fixatives are 4% paraformaldehyde (aldehyde fixative) and 70% ethyl alcohol (alcohol fixative). Paraformaldehyde can be used for antigen conjugation, and it is a good choice for immunofluorescence analysis. Ethyl alcohol can precipitate the protein with dehydration. The soluble component and lipid component may get lost and the cell would be permeabilized at the same time.
Methods of Immunofluorescence co-localization
Use two secondary antibodies, which are raised in different species and carry different fluorophores, to recognize the primary antibodies and bind to them. And And please use a secondary antibody that was raised against the species in which the primary was raised.
Why is the signal of the tissue section weak?
Because the antigen epitope may be covered. Uncompleted dewaxing of tissue section and unsuccessful antigen retrieval both may cause false-negative results.
What lead to high detecting background and how to fix them?
a, The autofluorescence. Measure: set a self-control detection to check it. b, The sealing is not good enough. It would bring high detecting cost, but you can also optimize the sealing ability to the situation. c, Dry slides. Measure: keep the sample moist in the process. d, There is a cross reaction between the secondary antibody and the sample. Measure: set a secondary antibody control group to detect it. e, In the whole process, especially after antibody incubation, it is crucial to clear the residual antibodies. Or it may cause high background.
Problems of weak signal detection and solution
a, Improper sample preservation, e.g. the fluorescence quenching or failing. To avoid this problem, please protect the sample from light and detect it in time. b, Unsuccessful target protein inducing (no expression or low expression the target protein ). To fix it, please try to optimize the related experimental procedure and set a positive control group. c, Use the wrong fluorescent group in detection so that the wavelength cannot be excited, and leads to weak or no signal.
How to choose lysate
Select mild lysate, e.g. NP-40 or TritonX-100 detergent to avoid denaturalization of the protein in the extraction process.
Protein A or Protein G?
Protein A and protein G are bacterial immunoglobin (IgG) binding proteins. While, protein A is originated from Staphylococcus aureus, protein G is of Streptococcal origin. Protein A has been shown to bind human IgG molecules containing IgG F(ab')2 fragments from the human VH3 gene family. In general, IgGs have a higher affinity for Protein G than for Protein A, and Protein G can bind IgG from a wider variety of species. The affinity of various IgG subclasses, especially from mouse and human, for Protein A varies more than for Protein G. Protein A can, therefore, be used to prepare isotypically pure IgG from some species. You can try Protein A/G in IP and ChIP experiments.
How to avoid the interference of light and heavy chain?
When detecting immunoprecipitates (or immunoprecipitated proteins) by Western blot analysis, the IgG heavy and light chains whose molecular weight are closed to that of the target protein may cause incorrect results. Replacing the secondary antibody with Protein A/G-HRP is a good way to reduce the interference of light chain. Replacing the secondary with the one that recognizes the light chain is a good method to reduce the effect of the heavy chain. Doing Western blot analysis with antibodies, which raised in different species from the antibodies in IP, is also a good choice.
How to choose antibodies in IP(Immunoprecipitation)?
Choosing a right antibody is very important for IP experiment. Fist of all, make sure that the antibody is validated with IP application. Secondly, antibodies for IP must keep unmodified. In addition, polyclonal antibodies are more suitable for IP. Polyclonal antibodies can recognize multiple binding sites, which would help to avoid false-negative results caused by antigenic epitope sealing. What’s more, polyclonal antibodies can connect (bridge) the antigenic epitope, enriching the protein and offering more FC locus for secondary reagents to amplify the signal detection.