Tetramer synthesis

Overview

The BRF MHC-Tetramer synthesis service provides custom synthesis of murine MHC Class I peptide tetramer reagents to researchers at ANU.  Tetramers and/or their derivatives are provided for research purposes only. Their use for commercial purposes is prohibited.

The amount of biotinylated monomer that is obtained is dependent on a number of factors.  One factor that determines the success of tetramer production is the binding affinity of the peptide for the MHC Class I molecule.  The yield of monomer will therefore be variable for each folding and will determine the number of aliquots available for subsequent tetramerizations.  Biotinylated monomer will be aliquoted and stored in 50ug units.

Tetramers are labeled with streptavidin-allophycocyanin (SA-APC) and streptavidin R-phycoerythrin (SA-PE).

Quality control

All tetramers are purified by size exclusion and ion exchange chromatography and each tetramer is tested for level of biotinylation.

Acknowledgement in publications

Please Note: All tetramers synthesised in the BRF must be acknowledged in the ”Materials and Methods” section of your paper and reference to use of the BirA enzyme in the synthesis MUST be cited, as shown below.


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

Applications

MHC Class I Alleles Available

H-2D(b)

H-2D(d)

H-2K(b)

H-2K(d)

H-2L(d)

Price

Price is based on the cost of the initial folding and the scale of tetramerization/label required.  Subsequent tetramerizations of aliquots of monomer from the same folding are available at a reduced price, provided no new monomer preparation is required.  Prices are based on our current costs to produce tetramers.

For example - if the folding yields 1000µg ( 5 x 200µg aliquots), the cost for the first prep will be the cost to produce biotinylated monomer plus labelling at the scale of your choice (currently 50, 100, 200µg).  Choice of label will also affect the cost.  For subsequent tetramerizations from stored monomer you will only pay for the labelling/tetramerization.

Please contact the BRF to enquire about price.

Phone: (612) 54326
Email: brf@anu.edu.au

Order form

Please complete a Tetramer Synthesis Order Form and discuss your requirements with the BRF beforeplacing your first order.

10mg of each peptide is required for each tetramer production.  Peptides should be HPLC purified and in powder form.  Analytical HPLC and MS should be performed on all peptides prior to use.  Ensure that your peptide is clearly labelled with your name and peptide ID. You will be informed by email when the tetramer is ready for pickup.

A tetramer order form can be downloaded.

Tetramer synthesis order form (PDF 63KB)

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