IgG - main low molecular weight immunoglobulin present in mammalian serum (75 % of serum immunoglobulins)
- Molecular mass ~150 to 170 kDa depending upon a species (In reduced conditions it is 50 and 24 kDa for mouse IgG)
- Molecular weight and isoelectric point of various animal immunoglobulins can be found here. Main properties of human immunoglobulins are described here.
- IgG constitutes of two light chains: 23 kDa each; two heavy chains:~50 kDa each (for H chain subclass ϒ1,2,4) and 60 kDa (ϒ3) connected by disulfide bonds
- 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 is 1.36 for a solution of 1 mg/ml (Johnstone and Thorpe 1988)
- Weight of IgG (or other protein) to molar quantity can be converted here
- 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
Protocol and description of nautralization of IgG with a protein or peptide can be found here.
Some interesting features of IgG antibodies:
- 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
Chosen examples of techniques where IgG antibodies have been successfully used:
- to prepare affinity columns and perform affinity purifications
- in Western Blot analysis
- in ELISA
- biotinylated or labelled with fluorescein or other chromophores
- in rocket immunoelectrophoresis
- in immunoprecipitation
- in immunogold labelling
- in immunohistochemistry
Agrisera Antibody Production Guide check here.
How to store IgG antibodies? Do antibodies last forever? (you might also want to check this link)
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.
Antibodies present in serum
This is a very stable format for antibody storage. In -20°C or -70°C serum can be usually stored for years. In some specific cases this time can be shorter for anti-peptide antibodies. For very short time periods serum can be stored at + 4°C. In some cases more careful freezing with a first step at -20°C followed by -70°C can 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 in this format are stable. They can be stored in -20°C or -70°C for years. For short-term use some azide to the final concentration of 0.02 % or other preservative.
Affinity purified antibodies (on a specific matrix allowing protein or peptide coupling)
Used to be the most unstable. Care should 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 abtibodies can precipitate directly after purification is completed if it is performed in the cold, while the activity may still remain. It is difficult to predict storage conditions for a given antibody in advance - there are some alternatives:
- -20°C or -80°C
- + 4°C with preservatives like azide (0.02%) or merthiolate
- -20°C with glycerol; Final concentration of glycerol 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-sterilised and aliquoted to avoid multiple freezing and thawing. Some antibodies cope well with thawing some might loose their activity.
- Storage at protein concentration around 0.5-1 mg/ml.
- Do not purify all serum you have at once. Antibodies in serum can be more stable compare to affinity purified material.
Important note: Sodium azide can inhibit horseradish peroxidase enzyme as well as interfere with some coupling methods and biological assays. However, its amount present in certain preparations as - 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:
- Thimerosal at 0,01%
- Gentamicin sulfate at 50 ug/ml
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.