NP40 or Triton as membrane protein detergent - (Apr/22/2010 )

Hi,
Im using ripa buffer to solubilize brain membrane protein.
I checked ripa components of others!, im wondering if NP40 is better than tritonX-100 for my case.
If someone knows about detergent efficient for membran protein solubilization, please, let me know
thanks

Hi Luciana,

check out the NP40 vs. TritonX-100 messy area of nomenclature.

Although NP40 is a good general detergent to solubilize membrane proteins, you often have to optimize the solubilization conditons. Detergent is only one factor. There's also time, temperature and the solution properties (salt, pH etc.).
In the end it depends, what you're planning to do with the extracted membrane protein sample. If the goal is to get the target protein onto a western you could go with 15%SDS (plus 5% dodecylmaltoside may be a good idea). If you want to carefully solubilize the target protein in its functional state, then you may need to optimize. This can be done with a screen of detergents and extraction conditions.
Check with Dilyx' solubilization screens - they'll be offering a membrane protein solubilization screen.

Cheers,
David

luciana on Apr 22 2010, 03:33 AM said:

Reusable medical instruments are required to be properly cleaned and disinfected between each use. Cleaning is defined as the removal of all foreign material such as blood, protein, cellular debris, tissue, respiratory secretions, mucus, saliva, feces, etc. from objects. For cleaning detergents and enzymes on aqueous medium are used . Enzymes enhance detergent cleaning by breaking down large, hard to remove materials into smaller, easy to remove fragments. There are three basic types of enzymes used in detergents: proteases, amylases, and lipases. Proteases are the most important type of enzyme to look for when choosing an enzymatic detergent for medical use because there is a high content of protein in most body fluids (including blood, tissue and mucous) which cannot be easily removed with regular detergents/surfactants and water. Proteases break down protein into individual amino acids or short strings of amino acids (peptides). Amino acids and peptides are much more soluble in water and will float away from surface or tubes of the instrument. Trypsin and Subtilisin are proteolitic enzyme that performing the characteristic of a enzymatic cleaning when set under suitable chemical conditions pH (about 7.5 - 8.5) and temperature (about 37 °C) that are those for their optimal operating.  Amylases are not essential components in enzymatic detergents because they have limited action on carbohydrates/starches that are very soluble in water and tend to be easy to remove with most detergents/surfactants and water. Lipases outside the body (in vitro) have limited effectiveness. Lipases are soluble in water and lipids are insoluble in water. The lipase has to mix with the lipid in order to break it up via hydrolysis. Since hydrolysis only occurs at the interface between the lipid droplet and the aqueous phase (water), this reaction is relatively slow and ineffective. Appropriate detergents/surfactants are more effective at removing lipids from medical instruments than lipases. Detergents must be non-corrosive and do not attack any metal or plastic surfaces on medical instruments. Properly formulated enzmatics will efficiently work in mild conditions and will not damage valves, rubber gaskets or any surface of a flexible fiberoptic endoscope or other medical instrument. Enzymes should also be fully biodegradable. Surfactants are surface active agents with wetting, detergent and emulsifying properties. Surfactants have both hydrophilic (water loving) and hydrophobic (water avoiding) properties and play a key role in soil removal. Since hydrophobic regions on a medical instrument surface can prevent a disinfectant from contacting and disinfecting the contaminated surface, it is important to add a carefully selected surfactant as a wetting agent to the solution. Another benefit of a good surfactant in an enzymatic detergent is that it will prevent protein fragments from redepositing on a medical instrument. Surfactants with good wetting properties will facilitate increased enzymatic action in an enzymatic detergent and NP-40 a nonionic polyoxyethylene surfactant that is most frequently used as a component of cell lysis buffers or other solutions intended to extract and solubilize proteins. The Dignam and Roeder nuclear extract procedure, widely used - see you - https://mylabo.files.wordpress.com/ , employ as detergent NP40 to confirm that is it the best for this application. I make a enzymatic cleaning solution place on board to Orphée Mythic 22 hematological analyzer with these specifications : dissolve 6.05 g/L Tris-base, 0.098 g/L (90 μl/L) 2-phenoxyethanol, 250 mg/L 2-chloroacetamide, 8.16 g/L NaCl, 1.98 g/L (1.98 ml/L) Tergitol NP-40, 10 mg/L Trypsin proteolytic enzyme, adjust pH to 8.50 with 0.45 ml of fuming HCl 37% concentrated and fill up to 1.0 liter with dH2O. To leave overnight at RT for to obtain a clear solution, add 0.4 ppm of formaldehyde and then filter on 0.45 μ filter.