Chromatography

Overview

Protein and antibody purification, and peptide mapping using chromatography

The facility is equipped to perform a wide range of liquid chromatography, with emphasis on the separation of biomolecules.  The combination of the Amersham-Pharmacia SMART System and ÄKTA Explorer allow for the delivery of precise solvent gradients at flow rates from as little as 10 mL /min to 100 mL/min over a wide range of back pressures.  Both systems provide programmable absorbance monitoring at three wavelengths simultaneously.

 

SMART System

The SMART system provides very high sensitivity with automated temperature control and sample collection.  It is ideally suited to analytical applications, for example, microbore (1mm i.d.) reversed-phase high performance liquid chromatography peptide mapping of post-translational modification sites.  The peptides so resolved may be taken to subsequent Edman peptide sequencing and mass spectrometry.  We have applied this approach many times to determine phosphorylation sites.  We have also employed this approach to the determination of acetylation, photoaffinity labelling and protein cross-linking sites.  Apart from reversed-phase columns a variety of ion-exchange, gel-permeation, hydrophobic interaction and affinity columns are available giving protein sensitivities in the ng range.

ÄKTA Explorer


The ÄKTA Explorer serves as a highly versatile preparative system as a result of its programmability. It is housed in a refrigerated cabinet thus obviating the need for the operator to work in a cold room.  It is configured with a dedicated programmable sample pump that provides effortless loading of large volumes.  This feature greatly simplifies procedures involving high volume samples, which include affinity purification of monoclonal antibodies from culture supernatants and Ni-NTA agarose affinity purification of secreted poly(histidyl) recombinant proteins.  A large variety of ion-exchange, gel-permeation, hydrophobic interaction and affinity columns are available for preparative applications.  Multiple runs may be programmed in advance and the system has buffer preparation capabilities that allow one to scout chromatographic variables.

More Information

An excellent resource for protein purification are a set of handbooks (9 in total) from AmershamPharmaciaBiotech that cover general guidelines and strategies for protein purification and different types of chromatography suitable for protein purification.

Protein purification handbook (PDF 1.63MB)

Affinity chromatography handbook (PDF 2.22MB)

Antibody purification handbook (PDF 1.51MB)

Hydrophobic interaction chromatography (PDF 496KB)

Ion exchange chromatography (PDF 933KB)

Reverse phase chromatography (PDF 488KB)

Recombinant protein handbook (PDF 2.05MB)

GST-fusion chromatography (PDF 1.54MB)

Gel filtration handbook (PDF 2.55MB)

 

Price

  Internal External Non-Profit External Commercial
Usage (per day) $75 $225 $225 + labour.
Consumables:

These charges apply to all users:

Column (Protein A or G)

Sulfolink columns

Tubes

Specialised buffers/reagents

Membrane filters

$215

$225

$0.15 each

$0.05/g

$1.65 each

 

Order form

A chromatography order form can be downloaded.

Chromatography order form (PDF 46KB)

Terms & conditions

General

  1. The BRF operates on a cost recovery basis.  The percentage of subsidization is determined by The John Curtin School of Medical Research.


  2. No samples or orders will be processed without an authorised* sample submission/order form and valid charge code.

 (*Authorised by signature of the PI/Lab Head, or by granting of electronic access to BRF ordering systems with the authority of the PI/Lab Head).
  3. It is a condition of the contract between the ACRF and JCSMR/ANU that "the Foundation (ACRF) is to be acknowledged in all scientific publications which utilise the Facility (BRF) at the Institute (JCSMR)". 
All work performed in the BRF, whether full service or not, and the use of any BRF resources should be acknowledged in all publications arising from that work. This should be in the “Material and Methods” section of the paper (see examples below). Any further individual acknowledgements are solely at your discretion.
  4. 

A reference to the publication should be sent to the Manager of the BRF once the paper is published (includes theses).
  5. 

BRF collaborations and co-authorship:  although the BRF is primarily a service unit, there are instances where BRF staff make significant contributions to either the technical or intellectual input of the project. This should be discussed with the staff member concerned prior to commencement of the collaboration. 



Consumables and data

  1. 

The customer agrees to cover the cost of any consumables ordered on their behalf if the customer fails to supply the correct quantity and quality of the starting material required for processing.  Details of the quality and quantity required are on the BRF website and sample submission form. 


  2. Customers are responsible for archiving data generated by the BRF. Data generated by the BRF are solely for the use of the customer and their collaborators.  Data is not to be sold to a third party. 

 The BRF shall not be responsible for data output generated from samples that deviate from recommended protocols as requested by the customer.


  3. Upon receipt of consumables and/or data, the customer accepts responsibility for the correct handling, use, storage and disposal of the consumables/data.
  4. The BRF extends no warranties of any kind in respect to the consumables.

  Any consumables sold may have hazardous properties.  The customer agrees to use appropriate caution and safeguards as not all properties are known. 


  5. Consumables sold by the BRF shall not be transferred to another party without the written consent of the BRF.  The BRF is not responsible for any losses arising from the use of consumables.  

The BRF will not be liable to the customer for any loss, claim or demand made by the customer due to acceptance, handling, use, storage or disposal of consumables and/or data by the customer, except to the extent permitted by law when it is the result of wilful misconduct on the part of the BRF or its employees.

Acknowledgement in publications

Examples

Real-time PCR


“Amplifications were performed in 384-well optical reaction plates (Applied Biosystems) with a 7900HT Fast Real-Time PCR System at the ACRF Biomolecular Resource Facility, The John Curtin School of Medical Research, Australian National University, using SDS 2.4 software to analyse raw data.”



DNA Sanger Sequencing


“Amplified PCR products were purified and sequenced on an AB 3730xl DNA Analyzer (at the ACRF Biomolecular Resource Facility, The John Curtin School of Medical Research, Australian National University) following the manufacturer's protocol (Applied Biosystems 2002).”

Peptide synthesis


“Peptides were synthesized chemically using the 9-fluorenylmethyloxycarbonyl (Fmoc) method on a CEM Microwave-assisted Peptide Synthesizer and purified by one round of C18 reversed-phase HPLC by the ACRF Biomolecular Resource Facility at the John Curtin School of Medical Research, Australian National University. As required, the N- or C-terminus, or both, were protected by acetylation or amidation, respectively.” 



Tetramer synthesis


“Cells were surface stained with APC-labelled tetramers consisting of murine class I MHC molecule (H-2Db), b2-microglobulin and influenza nucleoprotein peptide NP366–374. Tetramers were synthesised at the ACRF Biomolecular Resource Facility at The John Curtin School of Medical Research, Australian National University, using BirA enzyme synthesized as described (O’Callaghan et al., 1999). [O’Callaghan, C.A., Byford, M.F., Wyer, J.R., Willcox, B.E., Jakobsen, B.K., McMichael, A.J. and Bell, J.I (1999). BirA Enzyme: Production and Application in the study of membrane receptor-ligand interactions by site-specific biotinylation. Anal. Biochem. 266, 9-15.]”

Updated:  23 September 2017/Responsible Officer:  Director, ACRF/Page Contact:  Web Admin, ACRF