Thermo Scientific™ Nunc™ 1.8mL Externally-Threaded Universal Tubes and Racks

In all likelihood, your antibody stopped working because it lost its reactivity. This happens because antibodies (and most other proteins) are less stable at low concentrations (e.g., the µg/mL range and lower) than they are at higher concentrations. For example, proteins adsorb to surfaces like the walls of their containers due to charge-mediated and hydrophobic interactions. This occurs regardless of the protein concentration, and it usually results in some degree of protein denaturation and loss of activity. However, at low protein concentrations the impact of adsorption is larger per unit of time than at higher concentrations. Antibodies in solution also aggregate with each other for the same reasons that they adsorb to surfaces, sometimes resulting in loss of activity. How fast your diluted antibody loses activity in storage is unpredictable, so store your diluted antibodies no longer than overnight at 2-8 degrees C and then discard them. Better yet, make a fresh working dilution each time you need to use the antibody.

For most of the antibodies that we sell, a single freeze-thaw cycle should not damage them. Remove the antibody from the freezer and thaw it quickly. Mix it gently (do not vortex) and keep it on ice or in a refrigerator at 2-8 degrees C. It can be stored this way for approximately 1 month. If your working dilution of antibody got frozen, it should not be reused after it thaws. Antibodies in diluted solutions are not stable to freezing and thawing.

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.

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 techsupport@thermofisher.com if you have further questions about antibody stability.

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 degrees 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.

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.

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.).

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 techsupport@thermofisher.com. If Technical Support can positively identify the product, it's easy to determine its availability.

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).

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.

Please go to our Primary Antibody Selection Tool (https://www.thermofisher.com/order/genome-database/browse/antibody/sub-type/antibody_primary) 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.

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.

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 techsupport@thermofisher.com with the catalog and lot numbers.