Standard permeabilisation method (Triton X) appears to destroy antibody-carbohydrate antigen binding. Can anybody suggest any alternative methods of permeabilisation that may not cause this issue?
You can use other detergents that are less harsh (Tween-20). You can also use organic solvents, such as methanol and acetone
hi there,
Solvents:
1. Acetone fixation will also permeabilize
2. Methanol fixation can be used to permeablize but is not always suitable.
Detergents:
1. Triton or NP-40
Use 0.1 to 0.2% in PBS, 10 minutes only.
These will also partially dissolve the nuclear membrane and are therefore very suitable for nuclear antigen staining.
Note: as these are harsh detergents, they will disrupt proteins if they are used at higher concentrations or for longer amounts of time which will affect staining results.
2. Tween 20, Saponin, Digitonin and Leucoperm Use 0.2 to 0.5% for 10 to 30 minutes.
These are much milder membrane solubilizers. They will give large enough pores for antibodies to go through without dissolving plasma membrane. Suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane. Also suitable for soluble nuclear antigens.
Cytoskeletal, viral and some enzyme antigens usually give optimal results when fixed with acetone, ethanol or formaldehyde (high conc).
Antigens in cytomplasmic organelles and granules will require a fixation and permeabilization method depending on the antigen. The epitope needs to remain accessible.
hope this help