HPLC Gel Permeation Chromatography/Laser Light Scattering Determination of Native Protein Molecular Weights

HPLCSize Exclusion Chromatography/ Laser Light Scattering Determination of Native Protein Molecular Weights

The new service offers the exciting capability of determining the native molecular weights of proteins, their protein:protein and other macromolecular complexes. The MW determination depends only upon the downstream light scattering (LS) and refractive index (RI) detectors, and is thus completely independent of elution position.Hence, non-globular shape and/or possible interaction with the size exclusion chromatography (SEC) support, which otherwise pose severe limitations with regard to the use of SEC for estimating MW, have no impact on the molecular weights determined by LS. Based on analyzing 15 protein standards that range from 6.5 kDa to 457 kDa, native MW’s may be determined quickly and with an error of less than 10%.

SEC-LS system

The size exclusion chromatography is carried out on one of the following columns:

Superose 6 HR10/30 column (Pharmacia), which separates proteins with molecular weights that range from approximately 30 kDa to 2x10⁶ Da
Superdex 200 HR10/30 column (Pharmacia), which separates proteins with molecular weights that range from approximately10 to 600 kDa
Superdex 75 HR10/30 column (Pharmacia), which separates proteins with molecular weights that range from approximately 1 to 70 kDa.

The total column volume is ~24 ml and the standard buffer is 20 mM HEPES, 100 mM KCl, 1 mM EDTA with/without 1 mM DTT, pH 8.0. The SEC is carried out at a flow rate from 0.3 ml/min to 0.6 ml/min (depending on teh column used) at room temperature and the run time is about 1 hour. Although standard SEC runs are carried out in an "analytical mode" (without collection of the eluting peaks), fractions can be collected upon request and at an additional charge. Because of the extended equilibration time that is needed to establish a stable LS baseline and the additional standards that need to be analyzed after changing buffers, we encourage use of the standard HEPES buffer. Samples that must be subjected to SEC in other buffers will be subject to an additional charge and longer turn-around. The SEC column is connected on-line with the following HPLC flow detectors:

multiwavelength UV/VIS detector (Waters 996 Photodiode Array- PDA detector, Waters, Milford, MA)
LS detector (DAWN^®- DSP, Wyatt Technology, Santa Barbara, CA)

RI detector (OPTILAB DSP, Wyatt Technology, Santa Barbara, CA)

The system is calibrated using five proteins (cytochrome c, carbonic anhydrase, ovalbumin, bovine serum albumin, and alcohol dehydrogenase) that extend from 12.4 to 147 kDa. The MW is determined also from the absolute measurement of the scattered light intensity measured at 18 different angles . Although the UV signal does not enter directly into the MW calculation, comparison of the UV/RI signal ratio across the peaks of interest provide information relating to the homogeneity of the sample. Thus, if there are several peaks that arise from different oligomers of the same polypeptide, the UV/RI ratio should remain unchanged for all these peaks throughout the chromatogram. This value can be used also to independently estimate the extinction coefficient for the protein.

Sample requirements for one run:

Amount: 100-300 µg protein/run with the larger amounts being required as the MW is decreased below about 40 kDa
(click here for detailed table for sample requirements for proteins)

PLEASE NOTE: The amount indicated refers to FILTRATED sample injected onto the column.
CAUTION: Please note that all samples are filtered through a 0.22 µm filter (low binding Durapore® membrane, Millipore, Cat. No. UFC30GVNB) immediately prior to analysis. We strongly recommend that a trial filtration is performed before sample submission to avoid/account for sample loss during this procedure.

Note: The filtration is performed just before injecting the sample on the system. At this point the system is already equilibrated and calibrated at the conditions requested by the investigator. Hence, the set-up charge will be billed even for samples that failed to filter through the 0.22 µm filter (low binding Durapore® membrane, Millipore, Cat. No. UFC30GVNB) and subsequently were not injected/analyzed.

Volume: 250 µl or less is recommended; up to 750 µl may be loaded with some loss of resolution

Glycerol: less than 5%*

Please contact Ewa Folta-Stogniew at least 48 hours in advance before submitting samples (especially unstable proteins) to ensure system availability.

* samples can be submitted in buffer with higher glycerol concentrations and buffer exchange carried out prior to the analysis for an additional charge

For sample submission form go to HPLC SEC/Laser Light Scattering Sample Submission Page.
For pricing go toService Charges for HPLC SEC/Laser Light Scattering.

Introduction to Light Scattering	Light Scattering Theory	Light Scattering Data Set	Results for Standard Proteins	Light Scattering Service	Light Scattering Front Page