Anitta Denny

IMS12019
Purity and homogeneity check
Purity check:
As with water-soluble proteins, SDS-PAGE is the most widespread method for assessing the purity of
membrane proteins. Coomassie Blue, silver staining or Deep Purple is commonly used. The Laemmli
system is commonly used. Some modifications may be necessary for membrane proteins. Suitable
products are listed in the ordering information section.
Boiling of the sample with SDS can cause aggregation of membrane proteins. As an
alternative to boiling, incubation at 600C for 30 min or at 370C for 60 min are useful starting
points for preparing the sample for SDS-PAGE. On the other hand, some membrane proteins
are fully compatible with boiling and boiling may be required for complete solubilisation with
SDS.
Membrane protein frequently do not move according to the expected moleculr weight in SDSPAGE. They often move faster possibly due to incomplete unfolding or due to binding more
SDS per mass unit protein as compared with a water-soluble protein.
SDS-PAGE Clean-Up kit has successfully been used to treat samples containing interfering
detergent or that are too dilute for SDS-PAGE. With this kit, proteins are quantitatively
precipitated while interfering substances remain in solution.
Size homogeneity characterization:
Protein aggregation is a common issue with membrane proteins. Aggregation often appears to be
irreversible and it may occur slowly over time but also rapidly and unexpectedly with modest changes
in ionic strength, pH, protein:detergent ratio and other factors. For membrane proteins, it is as
important to keep track of aggregation as it is to monitor protein activity.
Aggregation may not always be detected by SDS-PAGE since SDS solubilises most aggregates. Gel
filtration is the method of choice for rapid detection of aggregation and it can be applied under a wide
variety of conditions. It is widely used as an efficient assay to assess the size homogeneity in purified
membrane protein samples.
Gel filtration allows detection of relatively small changes in the size of detergent-protein complexes
when different detergents are compared. Gel filtration is often used to give an indication of the
suitability of different detergents for a particular protein. Separation with gel filtration is thus an
important tool for qualifying the membrane protein preparation for further analysis. As an example,
gel filtration is very often used for assessing the suitability of different detergents for membrane
protein crystallization. In some cases, however, membrane proteins have been found to crystallize
under conditions where the protein preparation did not look homogenous
Charge homogeneity characterization:
High-resolution anion exchange chromatography is widely used for purification of membrane
proteins, often as a second chromatography step after IMAC. It is also an excellent technique
for charge homogeneity characterization of purified membrane proteins. Figure 1.14 shows

charge homogeneity characterization of fractions obtained from the IMAC purification

of histidine-tagged cytochrome bo3 ubiquinol oxidase, a membrane protein complex,
overexpressed in E. coli. The first IMAC fraction gave a shouldered peak on Mono Q (left).
Heterogeneity was confirmed by SDS-PAGE. The second IMAC fraction gave a single sharp,
symmetrical peak on Mono Q. Homogeneity was confirmed by SDS-PAGE which showed four
bands corresponding to the expected subunit molecular weights of 58 000, 33 000, 22 000,
and 17 000.

Conditioning
The purified membrane protein often has to be concentrated or transferred to a suitable environment
for further analysis. Common manipulations include concentration, desalting, buffer exchange,
detergent exchange, and detergent removal.

Can fibrous proteins be purified?

In an experiment which explores the differences in composition of the two types of fibrous proteins
which are Stratum Corneum fibrous protein (SCFP) and Hair fibrous protein(HFP), although they
have the same helical structure, SCFP was purified by dialysis against 0.1M Tris buffer, pH 7.0 and
collected by centrifugation.
In another experimental procedure, fibrous protein was isolated from the matrix region of calf hoof
and made to react with an antibody to hair fibrous protein. For this, fibrous proteins were purified
from the clear supernatant by precipitation at pH 7.0 and 6.0 .
There is no much literature available on fibrous protein purification, which means that purification of
fibrous proteins is a challenging task today. Hereby mentioning the few references for the above
denoted experiments:

Fibrous proteins of bovine hoof, HOWARD P. BADEN, M .D. AND JOSEPH KUBILUS, PH.D.
Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, U.S.A.

Comparison of stratum corneum and hair fibrous proteins, HowARD P. BADEN, M.D., NoRAH M
cGILVRAY, LoRETTA D . LEE, PH.D., LYNN BADEN, AND JOSEPH KUBILUS, PH.D.

Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, U.S.A.