Frequently Asked Questions (FAQs)-Find answers to the most commonly asked questions regarding our antibodies concentrations and purification. If you still have any other questions about our products, please see the FAQs page or contact us directly.
Generally, the optimal antibody concentration is 1 mg/mL. For concentration > 5 mg/mL or < 0.5 mg/mL, antibodies should be diluted / concentrated.
Usually, the antibody concentration can be found on the product datasheet. If this information is absent then please contact us directly or refer to the following:
We often use the A280 method to measure the antibody concentration. Namely, antibody concentration is measured by absorbance at OD280 nm. An extinction co-efficient of 1.43 is used i.e. a 1 mg/mL solution will give an OD reading of 1.43.
The optimal antibody concentration must be determined experimentally for each assay, which gives the best results with the minimum background. Usually, an ideal dilution ratio is determined by using a series of dilutions in a titration experiment. For example, if a product datasheet suggests using a 1:2000 dilution, it is recommended to make dilutions of 1:500, 1:1000, 1:2000, 1:4000 and 1:5000.
In addition, a titration experiment is performed by first selecting a fixed incubation time and then a series of experimental dilutions of the antibody. Note that each dilution ratio should be tested on the same type of sample in order to keep the same experimental conditions.
Various dilutions to use in each application from different sources of the antibody are recommended in the table below:
| Application | Tissue culture supernatant | Ascites | Whole antiserum | Purified antibody |
| WB/dot blot | 1/100 | 1/1000 | 1/500 | 1 µg/mL |
| IHC/ICC | Neat –1/10 | 1/100 | 1/50–1/100 | 5 µg/mL |
| EIA/ELISA | 1/1000 | 1/10000 | 1/500 | 0.1 µg/mL |
| FACS | 1/100 | 1/1000 | 1/500 | 1 µg/mL |
| IP | - | 1/100 | 1/50–1/100 | 1–10 µg/mL |
| Approximate IgG concentration estimate | 1-3 mg/mL | 5-10 mg/mL | 1-10 mg/mL | - |
Antibodies are widely used in the bio-conjugations, and the coupling efficiency is significantly affected by buffer condition due to the buffer or buffer additives can influence the conjugation efficiency.
Our antibodies are dissolved in different buffer forms such as PBS or PBS plus Sodium Azide. Thus, If your antibodies contain other buffer additives besides the PBS, we recommend your purify the antibodies before your conjugation projects. Additionally, antibodies in ascites fluid, serum or hybridoma culture media are also not suitable for conjugation.
The optimal choice of antibody purification method depends upon the class/subclass of the antibody, the species in which it was raised and the intended use of the antibodies. We often use protein A or protein G affinity chromatography, as well as protein L to purify antibodies that from bioreactors, supernatants, ascites, and serum from small (mL) to large volume (liters) purification. After antibody purifications finished, SDS-PAGE is used for a confirmation check.
Yes. We have accumulated many years of experience in antibody or protein purification. Our purification methods include (NH4)2SO4 salt precipitation, ion exchange and size exclusion chromatography, protein A, protein G and protein L affinity chromatography, and custom affinity chromatography for purification of antibody from serum, ascites, and culture supernatants. For more information about our antibody purification service, please view the Antibody & Protein Purification Service page.
Generally, the antigen concentration of 1 mg/mL or higher is preferred. The more concentrated antigen is fine. If your antigen has a concentration below 0.2 mg/mL, please tell us for an optimal solution.
If you are interested in our antibody purification service, please directly contact us and we will provide you a suitable purification option that will obtain the best balance between yield, purity and cost.
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