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Protein Chemistry Core Lab

Houston, Texas

Protein Core Lab
Protein Chemistry Core Laboratory
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MHC Tetramer Order General Guidelines

All requestors are required to complete the Tetramer Order Form and send requests via e-mail to lxwang@bcm.tmc.edu. You will be informed by e-mail when the tetramer is ready to pick up. Our location and contact information:

One Baylor Plaza
DeBakey Bldg, Room M917
Houston, TX 77030
Phone: 713-798-3918
Fax: 713-798-3700

Please direct technical inquiries or information about ordering status to Lily Wang at 713-798-3918 or lxwang@bcm.tmc.edu.

Peptide

Requestors incur the cost of peptide production and the peptide can be dropped at the location listed above. Requirements:

  • HPLC purified peptide;
  • Ten milligrams for each tetramer production;
  • Powder form.

We suggest requestors order the peptide from the Protein Chemistry Core Lab at Baylor to ensure the quality. Otherwise, HPLC chromatograph and mass spectrophotometric analysis for the peptide will be required. We will also offer pick-up service if the peptide is ordered from the Protein Chemistry Core.

Cost

The charge for each tetramer preparation is $550. The final product we provide is 1 vial of 0.2 mg MHC/peptide tetrameric reagent, which is enough for 200-400 assays. Any additional vial for the same tetramer will be only $150 each, if no new monomer preparation is required.

Since the fluorescence labeled tetramers can only be store at 4°C, their shelf-lives are limited to 6-12 months. For the interests of long-term studies, you can ask for multiple aliquots at smaller size, such as 4 of 0.05 mg or 2 of 0.1 mg ones. A freshly made tetramer aliquot will be provided upon your request each time.

One of the factors that determines the success of the tetramer production is the binding affinity of the peptide for the MHC class I molecule. Unfortunately, we can not accurately predict the binding properties of a peptide according to its sequence. We also do not have specific numbers that would represent a binding threshold for the peptide. Therefore, the failure of tetramer production happens occasionally. In that case, a second attempt for the MHC/peptide folding and chromatographic purification will be tried with no extra charge.

Fluorescent Label

All reagents are labeled with either commercially manufactured streptavidin-allophycocyanin (SA-APC) or streptavidin-phycoerythrin (SA-PE) fluorophores. These labels were chosen for use based on experience from several laboratories using tetramers. Some lot-to-lot variation in SA-PE has been observed, but should not seriously affect tetramer sensitivity or background if the staining suggestions (provided with the product) are followed. If other labeling reagents are desired, they must be provided by the requestor.

Monomeric soluble MHC/peptide is also available upon request.

ß2-microglobulin

We can provide MHC class I tetramers with either human or murine ß2-microglobulin. However, please note that we typically fold all tetramers of every species with human ß2-microglobulin. Use of murine ß2-microglobulin may reduce the stability of the reagent. Typically staining of murine CD8+ T-cells with tetramer is NOT affected by the use of human ß2-microglobulin.

Available MHC Alleles

Please refer to the MHC tetramer lab page for the current available MHC alleles at the core. If the requested MHC allele is not in the table or if there are other requirements, such as a specific ß2-microglobulin needed to reconstitute class I of an unusual species, the requestor must supply the cDNA. We will perform the subcloning into the appropriate vector for protein expression and additional charges may apply. All expression plasmids containing MHC alleles will be retained at the core and will be used for future production and distribution of tetramer reagents.

Quality Control

Quality control of tetramer reagents produced includes the following:

  • All tetramers will be purified by size exclusion and ion exchange chromatography. FPLC chromatograms of the reagent demonstrating proper folding and multimerization will be recorded.
  • Each tetramer is tested for the level of biotinylation.
  • ELISA using anti ß2-microglobulin antibodies specific for properly folded MHC I.
  • Training in using the reagents can also be provided upon request.