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Your Position: Home > FAQ > FAQ—Solarbio Antibody
FAQ—Solarbio Antibody

FAQ Documentation—Antibodys


Q01:What is the difference between polyclonal and monoclonal antibodies?
A01:Polyclonal antibodies are a mixture of multiple antibodies targeting multiple antigenic epitopes produced by multiple B-cell clones.
Monoclonal antibodies are highly homogeneous antibodies produced by a single B-cell clone, specific to a particular epitope.
Q02:What are the advantages of polyclonal antibodies over monoclonal antibodies?
A02:a) Polyclonal antibodies can help amplify signals from target proteins that are expressed at low levels. They can bind to multiple epitopes on the target protein, allowing for the combination with more than one antibody molecule. However, this can adversely affect quantitative experiments, leading to inaccurate results.
b) Polyclonal antibodies can recognize multiple epitopes, which can lead to better results in IP/ChIP experiments.
c) Polyclonal antibodies can recognize proteins with high homology to the immunogenic protein, or screen for target proteins from tissue samples of species other than the immunogen source. For example, when the antigenic properties of an untested species are unknown, polyclonal antibodies can be used.
d) Polyclonal antibodies can recognize multiple structural domains and are capable of identifying antigens even when minor changes occur in the spatial structure or sequence of the original antigen, or when there are slight variations in some antigenic determinants. Typically, polyclonal antibodies are the preferred choice for detecting denatured proteins.
Q03:What are the disadvantages of polyclonal antibodies compared to monoclonal antibodies?
A03:a) Polyclonal antibodies are more prone to batch-to-batch variability.
b) Polyclonal antibodies are more likely to produce non-specific antibodies, which may generate background signals in some applications.
Q04:How to choose between monoclonal and polyclonal antibodies?
A04:When high specificity of the antibody is required, large quantities are needed, or consistent antibodies are necessary for long-term use, and the applications for the prepared antibodies are diverse, monoclonal antibodies are generally chosen. When the specificity of the antibody is not highly required, and the need is for detection experiments involving precipitation and agglutination reactions or for ELISA tests only, polyclonal antibodies are typically selected.
Q05:What is phosphorylated antibody?
A05:Phosphorylation antibodies:Antibodies prepared using a phosphorylated immunogen are referred to as phosphorylation antibodies.
Phosphorylation antibodies are used to detect the level of protein phosphorylation, focusing on understanding the activity state of the protein.
Q06:What is the difference between phosphorylated antibodies and non-phosphorylated antibodies?
A06:a) Phosphorylation antibodies are mainly prepared against phosphorylated antigens to detect changes in the protein's phosphorylation state (increase or decrease).
b) Non-phosphorylation antibodies mainly detect the expression of the protein in tissues or cells, its tissue distribution, or its cellular localization, and can also serve as an internal control for changes in phosphorylation levels.
c) Phosphorylation antibodies detect proteins that are phosphorylated and are designed specifically for phosphorylation sites, making them site-specific antibodies.
d) Phosphorylated proteins are often transcription-related factors. When using phosphorylation antibodies and regular antibodies to detect the same protein, in situ detection may reveal different cellular localizations. After phosphorylation, transcription factors move from the cytoplasm into the nucleus.
Q07:How to distinguish between reactivity and host species?
A07:The reactivity indicated in the antibody manual refers to the species that the antibody can react with; that is, the antibody can be used to detect samples from these species. For example, if a primary antibody has reactivity with Human, Mouse, and Rat, then this antibody can be used to test samples from humans and both small and large rats.
The host refers to the species from which the antibody is produced, which is the source of the antibody. When selecting a secondary antibody, it is important to pay attention to the source species of the antibody.
Q08:How to correctly choose a primary antibody?
A08:a) Determine the species of the samples used in your experiment, such as Human, Mouse, Rat, Pig, Goat, etc. Choose an antibody that is the same species or has reactivity.
b) Determine the type of experiment you are conducting, such as Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Flow Cytometry (FCM), or Enzyme-Linked Immunosorbent Assay (ELISA). Generally, the antibody manual or official website information will list which types of analyses the antibody is suitable for. You can choose an antibody that matches your application.
c) Analyze and understand the structural properties of the sample protein. Antibodies are prepared from various immunogens that immunize the host. Immunogens include：full-length proteins, protein fragments, peptides, whole organisms (such as bacteria), or cells. The antibody manual generally describes the immunogen. If you plan to detect a protein fragment, a special isomer, or a certain region of the full-length protein, you must choose an antibody prepared with an immunogen containing this fragment or domain.
Q09:How to choose a secondary antibody based on a primary antibodies?
A09:a) Identify the species of origin for the primary antibody; the reactive species of the secondary antibody should match the species of the primary antibody. For example, if the primary antibody is of mouse origin, choose a secondary antibody that is anti-mouse (such as sheep anti-mouse, rabbit anti-mouse).
b) In addition to matching the species of origin of the primary antibody, the secondary antibody must also match the class or subclass of the primary antibody. Polyclonal antibodies are generally of the IgG type, and a secondary antibody can be chosen that is anti-mouse IgG (sheep anti-mouse IgG, rabbit anti-mouse IgG). Monoclonal antibodies are more complex, as they have different classes and subtypes, so the corresponding secondary antibody must also be selected based on the subtype of the antibody.
c) Conjugation markers for secondary antibodies:Generally, the common conjugation markers for secondary antibodies include horseradish peroxidase (HRP), alkaline phosphatase (AP), fluorescent groups (FITC, APC, PE), and biotin. The choice of which probe to use for secondary antibody conjugation mainly depends on the specific experiment. For Western blot, IHC, and ELISA, enzyme conjugated secondary antibodies are commonly used, while immunofluorescence and flow cytometry typically use secondary antibodies conjugated with fluorescent groups.
d) Choosing the form of the secondary antibody
The entire IgG molecule:This type of antibody is suitable for most situations.
Fab fragments:They have only one binding site and are generally used to block endogenous immunoglobulins.
F(ab')2 fragments:These antibodies are composed of two Fab fragments linked by disulfide bonds for antigen binding. They are used in specific situations, such as when it is necessary to avoid the antibody binding to cells that have Fc receptors.
Q10:How to determine the dilution concentration of antibodies?
A10:The general practice is to follow the recommended dilution ratios provided in the antibody manual for experiments. However, due to various factors such as sample type, experimental conditions, and operating environment, the recommended concentrations should be used as a reference. Researchers need to conduct experiments with multiple concentration gradients around the recommended concentration to find the optimal dilution ratio. If the manual does not provide a recommended dilution ratio, a significant amount of preliminary experiments will be necessary to determine the best dilution ratio.
Q11:How to choose a positive control?
A11:a) Most antibody instructions provide test images, and the samples in the test images can be used as positive controls.
b) According to the provided Swiss protein number, positive controls can be found on the Uniprot website, which typically lists tissues expressing the protein.
c) Search for positive controls on The Human Protein Atlas website,where the expression of this protein in human tissues and cells can be found.
d) Search for literature and refer to cells or tissues expressing the protein in the literature as positive controls.
Q12:Can antibodies be used for species or applications other than those clearly indicated?
A12:The reactivity and application of a specific antibody that is clearly labeled are generally validated.It is uncertain whether other species and applications can be detected.
For unverified species, the homology between the antigen sequence of the antibody and the sequence of the protein to be detected can be compared. If the homology exceeds 85%, the possibility of the antibody detecting your target protein is relatively high, but even if it is high,it does not guarantee successful detection.
Q13:How to preserve antibodies?
A13:Antibodies need to be transported at low temperatures. After receiving the antibodies, they should be stored at -20 ℃ or -80 ℃, and some conjugated antibodies should be stored at 2-8 ℃. It is best to avoid repeated freeze-thaw cycles and store them separately. Separate packaging can minimize damage to antibodies caused by freeze-thaw cycles. Aliquoting should not be less than 10 μL per portion, as the smaller the volume of the aliquot, the greater the impact of evaporation and adsorption by the tube walls on the antibody concentration.
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