Detergent concentration

This article is about how to measure detergent concentration after concentrating a membrane protein sample.

1. The simplest way to control the detergent concentration is to use a higher cut-off concentrator if your protein plus detergent micelle is large enough. Michael Matho: “a 50kDa cutoff withheld a lot of detergent during concentration process and consequently your final concentration might increase significantly. For example we started with 0.25% DES and noticed increases of above 1%. This did not happen when using a 100kDa cutoff, and DES concentration remain pretty much constant.” It is easy enough to test the “maximal cutoff you can use w/o loosing your membrane protein in the flow through”.

2. Patrick Loll, Edward A. Berry and John K. Lee suggested TLC, which seems to have the least requirement for equipments -- “silica gel TLC plates and a chromatography jar”.

Patrick: “We've done this as an exercise in NSLS Membrane Protein Crystallization workshop for a few years, and it works like a charm. You can stain in a warm iodine chamber and visualize by scanning the TLC plate on a garden variety scanner (we use an inexpensive Canon LIDE that probably cost less than USD 60 five years ago). We quantify the spot intensity with NIH Image or equivalent, and get lovely linearity down to the CMC, spotting only 1 uL of sample--so we haven't seen any need to concentrate.

Edward: “spotting on a TLC plate and running beside standard amounts of the same detergent. From intensity/size of the detergent spot after developing you can bracket the detergent concentration. (And by the way they found that detergents are concentrated by ultrafiltration). To increase sensitivity, speedvac a volume too large to spot on the plate, dissolve the residue in MeOH.”

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins. Laura R. Eriks, June A. Mayor, and Ronald S. Kaplan. Analytical Biochemistry 323 (2003) 234–241

3. For sugar-based detergents (maltosides and glucosides), one can use some traditional chemistry to measure the sugar.

Bert Van Den Berg: “do a fehling-type assay”

Zhenfeng Liu: ”phenol-sulfuric acid reaction for quantification of sugars.” Biochemistry, vol 36, no. 19, 1997, p. 5887

Hari Jayaram: “sulfuric acid and phenol followed by Absorption measurement; using a standard curve against the same detergent ”. Anal Biochem. 2005 Jan 1;336(1):117-24. A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research. Urbani A, Warne T.

4. Christopher Law: Use surface tension properties and look at the drop shape (measure contract angle). “A small droplet of the detergent solution is deposited on a piece of Parafilm M and side views are recorded by two orthogonally arranged TV cameras.”

A Novel Method for Detergent Concentration Determination. Biophys J. 2006 January 1; 90(1): 310–317. Thomas C. Kaufmann, Andreas Engel, and Hervé-W. Rémigy.

5. Ezra Peisach: by Refractive index. “Refractive index measurements were performed using an OPTILAB DSP instrument (Wyatt Technology) with a P10 cell.”

Refractive index-based determination of detergent concentration and its application to the study of membrane proteins Pavel Strop and Axel T. Brunger. Protein Sci. 2005 August; 14(8): 2207–2211.

6. Michael Matho: NMR is the most accurate method “using a high detergent concentration stock solution you can assign resonance peaks to your detergent molecule bonds. Then you can set up a standard curve using different known detergent concentrations (for example from 10% down to 0.1%) by calculating the surface of your peak(s) which is directly related to your detergent concentration. Each time you need to know the concentration of a new sample, you just need to record the peaks, and use the three-click rule to deduct the unknown value.”

7. David Veesler and Kornelius Zeth suggested ATR-FTIR (Fourier transform infrared spectroscopy). “very accurate, fast (10min) and requires as low as 10uL of protein sample.”

PVeesler, D. et al. Production and biophysical characterization of the CorA transporter from M. mazei. Analytical Biochem. (2009). 388 :115-121.

8. Philipp Ellinger recommended a nice new method that uses a fluorescence dye, Hoechst 33342, to detect micelle formation. The method is fast and easy, can be adapted to high throughput, and is not limited to sugar-based detergents.

Jumpertz T, et al. High-throughput evaluation of the critical micelle concentration of detergents. Anal Biochem. 2011, 408(1):64-70.