IgG is the main low molecular weight immunoglobulin present in mammalian serum, and makes up 75% of serum immunoglobulins. Below are some quick facts about IgG.
- Molecular mass: ~150 to 170 kDa depending on the species. IgG consists of two light chains and two heavy chains connected by disulfide bonds. The light chains have a molecular weight of around 23 kDa each, while the heavy chains are around 50 kDa each for H chain subclass ϒ1,2,4, and 60 kDa for ϒ3.
- Isoelectric point 6.1-8.5 (7.3 +/-1.2) if monoclonal (polyclonal antibodies do not have a set isoelectric point as they are a pool of various classes).
- Isoelectric point 6.1-8.5 (7.3 +/-1.2), polyclonal antibodies do not have a set isoelectric point as they are a pool of various classes
- Concentration in serum: 10-20 mg/ml
- Glycosylation (by weight): 3%
- Extinction coefficient at 280 nm: 1.36 for a solution of 1 mg/ml, Johnstone, A. and Thorpe, R. (1988) “Immunoassays”. In: Johnstone, A. and Thorpe, R., Eds., Immunochemistry in Practice, 2th Edition, Blackwell Scientific Pub, London, 257-260.
- IgG molecule dimensions: 13.7 nm (width), 8.4 nm (height), Horng et al. 2008
- Distribution in a body: intra and extravascular
- Immunological function: secondary response
- Can self-assemble into hexamers, which form two dimensional crystals in aqueous solution (Shinichiro et al. 2014, Nature Materials)
- Activate mammalian complements
- Bind to Protein A or Protein G
Weight of IgG (and other proteins) to molar quantity can be converted here
Molecular weight and isoelectric point of various animal immunoglobulins can be found here.
Main properties of human immunoglobulins are described here.
Protocol and description of neutralization of IgG with a protein or peptide can be found here.
Examples of techniques where IgG antibodies have been successfully used:
- Preparation of affinity columns and affinity purifications
- Western Blot analysis
- Biotinylation or labelling with fluorescein or other chromophores
- Rocket immunoelectrophoresis
- Immunogold labelling
Agrisera Secondary Antibodies bind to: bovine IgG | cat IgG |dog IgG | goat IgG | hamster IgG | LLama IgG | rabbit IgG | sheep IgG | donkey IgG | guinea pig IgG | human IgA | human IgE | human IgG | human IgM | mouse immunoglobulins | rat IgG
Agrisera Antibody Production Guide check here.
Storage of IgG antibodies
Antibodies present in serum is a very stable format for antibody storage. In -20°C or -70°C serum can usually be stored for years, but in some specific cases, this time may be shorter for anti-peptide antibodies. For very short time periods, serum may be stored at +4°C and in some cases, more careful freezing with a first step at -20°C followed by -70°C may be beneficial. Some antibodies will store well as lyophilized serum at -20°C.
Total IgG fraction (IgG antibodies purified on Protein G matrix)
Generally, antibodies purified on a Protein G matrix are stable and can be stored in -20°C or -70°C for years. For short-term storage, use azide to the final concentration of 0.02%, or some other preservatives.
Affinity purified antibodies (on a specific matrix allowing protein or peptide coupling)
Antibodies that have been affinity purified are usually the most unstable. Care should thus be taken when considering storing conditions, which should be checked experimentally for every single antibody. Affinity purified antibodies against different epitopes can vary in stability. Some purified antibodies can precipitate directly after purification is completed, if performed in cold conditions, while the activity may still remain. It is difficult to predict storage conditions for a given antibody in advance. Below are some alternatives:
- -20°C or -80°C
- + 4°C with preservatives like azide (0.02%) or merthiolate
- -20°C with glycerol at a final concentration of 10% or 50%
- -20°C with BSA at final concentration of 0.05-0.5%
- Larger volumes of affinity purified antibodies are recommended to be filter-sterilized and aliquoted to avoid multiple freezing and thawing. Some antibodies cope well with thawing some might lose their activity.
- The protein concentration during storage should be around 0.5-1 mg/ml.
- Do not purify an entire volume of serum at once. Antibodies in serum can be more stable compare to affinity purified material, and the antibody can therefore benefit from being stored in serum form when not used.
Important note: Sodium azide can inhibit horseradish peroxidase enzyme, as well as interfere with some coupling methods and biological assays. However, the amount of sodium azid present in certain preparations (0.02 %) will be washed away in ELISA or Western Blot if a primary antibody is used at a dilution of at least 1:2000. Alternative agents for preventing bacterial growth of an antibody solution are:
- Thimerosal at 0.01%
- Gentamicin sulfate at 50 ug/ml
Note: please keep in mind that each antibody is different, and conditions and protocols applied for one antibody are not necessarily going to work for another antibody.
Recommended literature about IgG antibodies:
- Using Antibodies: A Laboratory Manual, E.Harlow and D.Lane, 1999,
ISBN: 0879695447; Publisher: Cold Spring Harbor Laboratory Press.
- Immunochemistry in Practice, A.Johnstone and R.Thorpe, 1988,
ISBN:0-632-01723-6; Publisher: Blackwell Scientific Publications.