Antibodies Support—Getting Started
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The concentration of most of our antibody products will be listed on the product label and in the manual. For primary and secondary antibodies, the concentration ranges from 0.1 to 2 mg/mL, depending on the product. The concentration range for most “prediluted” antibodies is 1–10 μg/mL; for what we refer to as “concentrates”, it is usually 50–500 μg/mL. For some products, we do not provide the concentration—these are packed by volume (e.g., 100 μL) or other unit amounts (e.g., 10 blots). Also, the concentrations of many former Zymed™ pathology antibodies (Cat. No. starting with 08, 18, or 28) are not provided. A few of our antibodies consist of unfractionated serum, concentrated hybridoma supernatant, or samples of ascites fluid. In these cases, the antibody concentrations are unknown. Please follow our dilution recommendations for these products.
The concentrations of antibodies packed by volume are not trade secrets, but the unit size is based on activity titration rather than antibody concentration. Thus, there is lot-to-lot variability in the antibody concentration. For most antibodies sold by volume or other amounts, the number of slides or western blots that you can stain with each vial from each lot does not change because the recommended dilutions remain the same. If you don’t know the concentration of your antibody, please contact Technical Support at firstname.lastname@example.org with the catalog and lot numbers.
Diluting an antibody 1:200 means that you add 1 volume of it to 199 volumes of a diluent (1 + 199 = 200). Similarly, a 1:1,000 dilution means that you add 1 volume of antibody to 999 volumes of the diluent (1 + 999 = 1,000). Let’s say that you need 20 mL of your antibody at a dilution of 1:200. It would be very easy to take 1 mL of antibody and add it to 199 mL of diluent. However, you only need a total of 20 mL of diluted antibody, so you can take 0.1 mL (100 μL) of antibody and add it to 19.9 mL of diluent (0.1 + 19.9 = 20). This is still a 1:200 dilution. If you only need 2 mL of diluted antibody, the mixture would be 0.01 mL (10 μL) of antibody added to 1.99 mL of diluent (0.01 + 1.99 = 2). This is also a 1:200 dilution. Whenever you dilute an antibody, mix it gently to ensure a homogeneous solution. We recommend against using a vortex mixer, as vortexing may contribute to inactivation of the antibody.
We normally do not disclose the amino acid (aa) sequences of our immunogens, nor do we provide any other specific information about them. The immunogen-related information that we do provide is listed in the manual for the antibody. We have chosen not to reveal the peptide sequences for most of our antibody immunogens, primarily because we do not seek patent protection for them. However, Technical Support can provide an approximate location of the immunogen in the protein (e.g., between aa 23 and aa 100), the general location of the epitope (e.g., C- or N-terminus, cytoplasmic or membrane domain), and other related information, if available.
Please go to our Primary Antibody Selection Tool and then select “Monoclonal” or “Recombinant” under “Antibody Type” on the left navigation pane, for our mouse and other monoclonal antibodies, or our recombinant ABfinity™ rabbit monoclonal antibodies.
Monoclonal antibodies are produced in mice, rats, hamsters, and rabbits. First, the host animal is injected with an antigen to initiate a humoral immune response. In most procedures, spleen cells from these hosts are fused in vitro with cultured malignant myeloma cells. The cell clones that survive the fusion step are known as hybridomas. Hybridomas are immortal because of their myeloma characteristics, and they are easily propagated in culture. Because of their B cell properties, some hybridoma clones continually synthesize and secrete a single, genetically homogeneous type of antibody, the monoclonal antibody. Monoclonal antibodies are therefore homologous to natural immunoglobulins from mice, rats, hamsters, or rabbits, but they can be produced by hybridomas in vitro, indefinitely. Mouse-, rat-, hamster-, and rabbit-derived hybridomas are currently the most common sources of monoclonal antibodies, and we offer monoclonals from these species that recognize a huge selection of antigens.
In general, for IgG antibodies, the heavy chain is approximately 55 kDa and the light chain is approximately 25 kDa.
Polyclonal antibodies are mixtures of related antibodies harvested from the serum of previously immunized animals. Serum-derived polyclonal antibodies can be produced in virtually any mammal. However, most polyclonal antibodies are produced in animals with large blood volumes like goats, rabbits, donkeys, and sheep.
An alternative source of polyclonal antibodies is egg yolk from birds like chickens and turkeys that have been immunized with antigens. Polyclonal antibodies isolated from egg yolk are of the IgY class. The IgY antibodies are functionally equivalent to IgG antibodies from mammals.
Most of our polyclonal antibody products are derived from goat or rabbit serum. We offer many polyclonal antibodies that recognize numerous protein antigens, including phosphorylated targets. Among our polyclonal antibodies are class- and species-specific antibodies (e.g., those that recognize IgG from a particular species), so they are used as secondary antibodies for immunodetection.
The abbreviation PAD stands for "Polyclonal Antibody Designation". The PAD is analogous to the clone name or number for monoclonal antibodies, but it is used as a unique identifier for polyclonal antibodies. The PAD is part of an agreed-upon nomenclature system that was developed so that researchers can cite antibodies in publications without using catalog numbers.
Yes, a few of our unconjugated antibodies can be purchased in 500 μL volumes. The catalog numbers are: R950-CUS (anti-Myc antibody) and R960-CUS (anti-V5 antibody).
Many of the antibodies that were sold by Zymed, Biosource, and Caltag are still available from us. In most cases, the same catalog numbers are still being used. In fact, the same production methods developed by these original suppliers are still used as well. This means that the antibody specifications, formulations, concentrations, usage guidelines, and the results you are accustomed to are the same now as they were in the past. The antibodies that have been discontinued were eliminated for 3 basic reasons: 1) we did not want multiple brand names on the same antibody; 2) we could no longer assure a reliable supply of the products; or 3) some antibodies simply sold too few units per year for us to continue to sell them. When you find a Zymed, Biosource, or Caltag antibody cited in the literature and you’re not sure if it’s still available, please contact Technical Support at email@example.com. If Technical Support can positively identify the product, it’s easy to determine its availability.
Secondary antibodies are directed against the species of the primary antibody. Therefore, you will need a secondary antibody that is raised in a species different from the host species of the primary antibody. For example, if your primary antibody is raised in a rabbit, you will need an anti-rabbit secondary antibody raised in a host species other than rabbit (e.g., goat, mouse, etc.).
Secondary antibodies may be either too specific (e.g., recognize only one host species of primary antibody) or too general (e.g., recognize whole IgG and any fragments thereof). In most cases, these limitations can be overcome by carefully designing the experimental system and choosing the appropriate secondary antibody. The following considerations are useful to help choose a secondary antibody:
- Determine the host species of the primary antibody (e.g., mouse anti-tubulin).
- Select an appropriate host species for the secondary antibody—you will need a secondary antibody that is raised in a species different from the host species of the primary antibody (e.g., goat anti–mouse IgG).
- Consider cross-reactivity or specificity issues of the secondary antibody.
- Highly cross-absorbed—for multiple-labeling applications or when using samples with endogenous antibodies
- Specificity—binds to correct fragments, classes, or chains of the primary antibody
- Use an appropriate detection or purification method.
- Label—appropriately conjugated to the correct enzyme, tag, or fluorophore for the chosen detection method
- Ability to bind to Protein A, Protein G, or Protein L—make sure the secondary antibody chosen has sufficient affinity for the molecules used upstream or downstream (i.e., Protein A–coated microplates)
- Consider requirements of the supplied secondary antibody.
- Supplied state—sterile liquid or lyophilized, suspended in PBS or Tris buffer, contains carrier proteins such as gelatin or albumin or the addition of stabilizers such as sucrose or microbial inhibitors
The recommended amounts of antibody to use are based on the protein concentration of the cell or tissue lysate. Also, amounts vary depending on the antibody source, as follows: for each 200–500 μg of cell or tissue lysate protein, we suggest adding 1–10 μg of purified monoclonal or polyclonal antibody, 1–5 µL of an unpurified polyclonal antiserum, 0.2–1 µL of ascites fluid, or 20–100 μL of hybridoma supernatant. These suggestions are guidelines only, so you will probably have to optimize your IP reactions empirically to determine the amount of antibody that provides the best yield and lowest background. Most likely you will do this by performing SDS-PAGE followed by western blotting of the immunoprecipitates and staining with antigen-specific antibodies.
It is useful to remember that an antibody will probably work in IP if it works well in immunohistochemical or immunofluorescence staining. This is because these applications depend on antibody recognition of the antigen in the native protein. The only way to be certain is to try the antibody in IP using the guidelines discussed above as a starting point.
Antibody Storage and Stability
Follow the storage recommendations provided in the manual and on the product label. Concentrated antibodies can be stored at 2–8°C for up to 1 month. For long-term storage, we recommend that all antibodies be aliquoted into single-use amounts and stored at –20°C in a non–frost-free freezer or at –80°C. Avoid multiple freeze-thaw cycles, as this will diminish antibody activity. Do not freeze diluted antibodies. Make the final working dilution just prior to use. It is possible to add stabilizing agents like glycerol (e.g., 40–50% (v/v)) to the antibody. However, unless we say that it is okay to do so, we can’t guarantee the performance of the product with stabilizing agents added.
Non–frost-free freezers are recommended for antibody storage because they do not go through automatic defrost cycles. To keep a freezer compartment frost-free, the freezer actually defrosts itself periodically with heating coils. Temperatures inside the freezer can rise to 0°C and above during the defrost cycle. If antibodies are subjected to these temperatures for a long enough time, they can undergo a partial thaw. This happens especially at the interface between the air in the sample container and the surface of the frozen contents. Since air warms faster than water, proteins at the interface are affected more than those in the depths of the tubes.
Even if you are using a non–frost-free freezer, you should pay attention to where your antibodies are stored inside it. It is best to store them in an area of the freezer compartment that experiences the least temperature fluctuation, which is usually in the center in the back of the freezer. Do not store antibodies near the front of the freezer compartment or on a shelf in the freezer door, where they are exposed to room temperature every time the freezer door is opened. Make sure as well that the antibody container is tightly sealed. This can help to prevent water loss from the antibody via sublimation (conversion of ice directly to water vapor that escapes from the containers). This slow process is basically freeze-drying at normal atmospheric pressure, and it can lead to damage to the antibody.
Generally, antibodies can be left at room temperature for up to a week without loss of activity. Hence, we ship many of our antibodies at ambient temperature. However, longer storage at room temperature or higher is not recommended, and we can’t guarantee the performance of the antibody under such circumstances. Please contact Technical Support at firstname.lastname@example.org if you have further questions about antibody stability.
Bovine serum albumin (BSA) is a protein-stabilizing agent, and sodium azide is a preservative that prevents bacterial growth. The carrier protein and preservative are added to increase functionality and longevity of the antibodies. Antibodies supplied with BSA and azide can be stored at 2–4°C.
HRP is inhibited by sodium azide, so our HRP-conjugated antibodies do not contain this agent. Our unconjugated antibodies are, however, supplied in solutions containing sodium azide.
We recommend that hydrophobic residues comprise 50% or less of all the residues in your sequence. Make sure there is at least one charged residue for every five amino acids: this is generally known to enhance the solubility of the peptide. Peptides (compared to polypeptides, which fold and bury the hydrophobic amino acids) are too small to fold, so just a few hydrophobic amino acids may leave them insoluble.
Other guidelines include:
- Peptides containing multiple Cys, Met, and Trp can be hard to synthesize.
- Some sequences are problematic in solid-phase peptide synthesis or cleavage and are best avoided; these include Asp-Pro sequences or stretches of amino acids that require bulky protecting groups on their side chains during synthesis.
- Glycine is often good for antigenicity as it has only a hydrogen side chain; this allows for complete rotation.
Most peptide antigens range in length from 12 to 16 residues and are relatively easy to synthesize. Peptides of 9 residues or shorter have been effective antigens for antibody production, but peptides longer than 16 amino acids may contain several epitopes and form secondary structures. Peptides in excess of 18 residues begin to present more synthetic challenges. Before you synthesize your peptide, we recommend doing a BLASTP search using your peptide sequence. This is to make sure that the peptide is not homologous or identical to a sequence in a completely unrelated protein in the host animal.
For Research Use Only. Not for use in diagnostic procedures.