Protocols

Plant organelle/membrane isolation

• Mitochondrial fraction
• Nuclear fraction
• Plasma membrane fraction
• Thylakoid extraction

Plant protein isolation

• Extraction of leaf proteins
• Diatom protein extraction
• Phenol protein extraction
• Protein extraction from grasses
• Ponceau membrane staining
• TCA acetone precipitation method

Western blot

• Western blot protocol
• Western blot video tutorial
• Peptide neutralization/competition assay
• Simultaneous Western blot
• Quantitative Western blot
• Quantitative Western blot video tutorial
• Western blot troubleshooting 
• Western blot using IgY

Other protocols

• Dot blot
• ELISA
• Immunoprecipitation
• Immunoprecipitation/IgY
• Immunohistochemistry
  
• Anti-KLH antibody removal 
• Yolk delipidation

Technical information

Antibody types

• IgG
• IgM
• IgY
• MW and pI for immunoglobulins

Purification

• Antibody purification
• Antibody purification - small amount of protein
• IgY purification methods
• Protein purification using antibodies
• Elution of antibodies from affinity columns

Other

• Antibody storage
• Antibody production guide
• Secondary antibody guide
• Problems with dialysis

Technical information > Molecular weight and isoelectric point of various animal immunoglobulins

Immunoglobulin	Approximate MW/ kDa (g·mol−1)	Number of subunits	pI*	A280 (mg/ml)
IgA, human	162	4	n.a.	1.34
IgE, human	190	4	n.a.	1.53
IgG, goat	144	4	n.a.	1.30
IgG, human	153	4	6.6 - 7.2	1.38
Subclass IgG1, human	146	4	8.6 ± 0.4	n.a.
Subclass IgG2, human	146	4	7.4 ± 0.6	n.a.
Subclass IgG3, human	170	4	8.3 ± 0.7	n.a.
Subclass IgG4, human	146	4	7.2 ± 0.8	n.a.
IgG, mouse	155	4	∼ 7.0	1.40
IgG, rabbit	150	4	6.5 - 7.5	1.36
IgM, human	950	20	5.5 - 7.4	1.33

*Isoelectric point (pI) values are theoretical calculations based on the distribution of charged amino acid residues in a protein sequence. Experimental conditions, such as buffer composition and pH, can also influence the behavior of proteins in solution. Beyond this, factors such as post-translational modifications and specific characteristics of the antibodies can also effect the pI. Therefore, precise determination of the pI for a particular protein often requires experimental techniques such as isoelectric focusing or capillary electrophoresis.

Polyclonal antibodies do not have a set isoelectric point as they are a mixture of various classes.

Mouse immunoglobulin subclasses: IgG1, IgG2a, IgG2b, IgG2c, IgG3
Rabbit immunoglobulin subclass: IgG (only one)
Rat immunoglobulin subclasses: IgG1, IgG2a, IgG2b, IgG2c

For more extensive information on different types of immunoglobulins, please visit the following pages: IgG, IgM, IgY

Average serum concentration of human IgGs (mg/ml):

• IgG1: 8
• IgG2: 4
• IgG3: 0.8
• IgG4: 0.4

Human immunoglobulins are composed of 82-96% proteins and 4-18% carbohydrates.

Antibodies to unconjugated human immunoglobulins

         


Immunoglobulins from various species in different purity grades