Frequently asked questions | ProImmune

Frequently Asked Questions

General Information

How long do you take to deliver?

Is there an additional charge for custom reagents compared to stock items?

Where can I find protocols for using your products?

Who are your distributors?

MHC Class I Pentamers

How do I order Pro5® MHC Pentamers?

What is the shelf-life for Pro5® MHC Class I Pentamers

What are the storage conditions for your Pentamers?

What is the concentration of your Pentamers?

Who provides the peptide for custom Pentamer synthesis, ProImmune or the customer?

How do you purify your Pentamers and what QC steps do you carry out?

Can you synthesize empty Pentamers?

What can I use as an isotype control for Pentamer staining?

Which antibodies do you recommend to be used with your Pentamers?

Do you have plans to produce rat, HLA-G or other unusual alleles?

Do you produce reagents for antigen-specific staining of CD4+ T cells?

CD1d Tetramer

Which alpha-Galactosyl-Ceramide(α-GC) should I use to load my CD1d tetramer?

How should I sonicate the lipid (specifically α-GC)?

How long will the loaded CD1d tetramer be stable for?

How long will the dissolved α-GC be stable for?

What should I use as a negative control?

Can human CD1d tetramers be used with mouse samples?

Do the human CD1d tetramers cross react with any other species?

Peptides

How should I dissolve my peptide?

How long will peptides be stable for once dissolved?

Can you supply me with lyophilized peptide only?

What is the minimum affinity for peptides used in custom synthesis?

 

How long do you take to deliver?

We usually ship stock items within 1-3 business days of receiving your order. Custom synthesized MHC-peptide Pentamer complexes are available approximately 6 weeks from ordering for standard synthesis or 12 working days for Rapid Synthesis. For further information click here.

Is there an additional charge for custom reagents compared to stock items?

Custom items are usually subject to a small set-up charge, which will be applied once for each item. The set-up charge will not be applied to repeat orders of the same specificity. For further information click here.

Where can I find protocols for using your products?

Please follow this link to our list of protocols.

Who are your distributors?

ProImmune sells products directly in most countries except as follows, where our products are sold exclusively through the following distributors.

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What is the shelf-life for Pro5 MHC Class I Pentamers?

All pre-labeled Pro5® MHC Pentamers are guaranteed for 6 months from delivery and unlabeled Pro5® MHC Pentamers are guaranteed for 12 months from delivery, when stored according to our instructions. ProVE® MHC Library Pentamers are guaranteed for 3 months.

What are the storage conditions for your Pentamers?

Fluorescently labeled MHC Pentamers should be stored at +2ºC to +6ºC, with the lid tightly closed, and should NOT be frozen; avoid prolonged exposure to light. Unlabeled MHC Pentamers should be stored frozen and aliquotted at -80ºC; avoid multiple freeze-thaw cycles.

What is the concentration of your Pentamers?

Our MHC Pentamers are sold as test quantities in PBS containing 1% BSA and 0.01% sodium azide. One test is sufficient material to stain 1–2 x 106 antigen-specific T cells. Labeled Pro5® MHC Pentamers are provided at a concentration of 0.05 mg/ml or 10µl/test. Unlabeled Pentamers are supplied at 0.5 mg/ml or 2µl/test.

Who provides the peptide for custom Pentamer synthesis, ProImmune or the customer?

ProImmune provides all peptides for the synthesis of Pro5® MHC Pentamers for quality control reasons. The cost of the peptide is included in the Pentamer list price.

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How do you purify your Pentamers and what QC steps do you carry out?

Every Pro5® Pentamer we ship has passed standard biochemical characterization plus two rounds of protein purification to homogeneity. In addition, we carry out conformational antibody testing on all samples to ensure the proteins are correctly refolded. This ensures that all ProImmune Pentamers are synthesized to the highest possible standard. We also test all batches of Pentamer by flow cytometry to ensure they are within acceptable limits of non-specific staining.

Can you synthesize empty Pentamers?

Empty MHC class I molecules are extremely unstable. It is therefore not possible to supply customers with ‘empty’ MHC molecules. However, you can purchase an HLA-A*0201 negative control Pentamer that is assembled with an irrelevant peptide antigen that is known to have no T cell response.

What can I use as an isotype control for Pentamer staining?

You should use another Pentamer, either of the same allelic restriction but containing a mis-matched peptide, or a mis-matched allele and mis-matched peptide combination. It is important, however, to ensure that your donor is serologically negative for this allele/peptide combination before using it as an isotype control.

Which antibodies do you recommend to be used with your Pentamers?

ProImmune sells a range of human and mouse antibodies that we recommend for use with our Pentamers. All of these antibodies have been tested alongside Pentamer staining to ensure compatibility. For anti-CD8 staining we recommend clones LT8 for human Pentamers and KT15 for murine Pentamers. If you would like to know about alternative suitable clones, please contact us.

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Do you have plans to produce rat, HLA-G or other unusual alleles?

At ProImmune we are keen to follow the specific needs of our customers. We now have available Mamu-A*01 and Mamu-A*02. View the full list of alleles available for custom synthesis. If you require the synthesis of alternative MHC molecules please contact us to express your interest.

Do you produce reagents for antigen-specific staining of CD4+ T cells?

Yes, ProM2 MHC class II monomers and ProT2MHC class II tetramers are now available as catalogue and custom reagents.

Which alpha-Galactosyl-Ceramide (α-GC) should I use to load my CD1d tetramer?

Funakoshi is the sole supplier of α-Galactosylceramide(KRN7000) world-wide and is licensed by Kirin Pharma Company Ltd.

(https://www.funakoshi.co.jp/export/product/KRN000001E.html)

How should I sonicate the lipid (specifically α-GC)?

Prepare no more than 500ul of a 0.2 mg/ml solution of lipid in PBS + 0.5% Tween-20. Incubate in a sonicating water bath (100W/20kHz) at 80oC for 2 minutes. After 2 minutes, check the solubility of the lipid and if not completely solubilized repeat the sonication step. Only attempt to load fully solubilized lipid to the tetramer. Incomplete solubilization will result in poor tetramer loading.

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How long will the loaded CD1d tetramer be stable for?

If stored at 4oC, the α-GC will be stable for at least 6 months.

How long will the dissolved α-GC be stable for?

If stored in aliquots at -80°C, the α-GC will be stable for over a year.

What should I use as a negative control?

ProImmune supplies a CD1d tetramer negative control.

Can the human CD1d tetramer be used with mouse samples?

The human CD1d tetramer is cross reactive with mouse samples. An example of the human tetramer used to stain splenocytes from a C57BL/6 mouse is shown here

Do the human CD1d tetramers cross react with any other species?

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The human CD1d tetramers will also cross react with non-human primates such as rhesus macaque monkey samples. The tetramers should be used following the same protocol as for human samples. For further information click here.

How should I dissolve my peptide?

The most appropriate solvent for your peptide will depend on the peptide sequence and the peptide application. For applications involving cell culture experiments peptides should NOT be dissolved in DMSO as this may interfere with downstream assays. For further information click here.

How long will peptides be stable for once dissolved?

Very hydrophobic residues (C, M and W) will only be stable for a week or two stored at -80°C once dissolved as they are prone to oxidation. Less hydrophobic peptides will be stable for several months stored at -80°C. Avoid freeze-thawing cycles as this will destabilise the peptide.

Can you supply me with lyophilized peptide only?

Yes, we can supply custom peptides to a variety of lengths, purities and modifications. We can also supply you with very cost-effective Matched Peptides in 1mg and 2mg quantities for our stock items, or if you concurrently place an order for custom MHC Pentamers of the same peptide sequence.

What is the minimum affinity for peptides used in custom synthesis?

As a general rule it is only possible to synthesize recombinant MHC complexes where the peptide has a medium to high affinity for the MHC allele. Although we do not exclusively rely on predictive algorithms, in our experience decisions based on binding affinities determined via these methods correlate well with the outcome of synthesis of custom MHC-peptide complexes. We therefore advise our customers to use the SYFPEITHI algorithm to score their peptides www.syfpeithi.de. We will typically only proceed with a custom synthesis for peptides scoring 21 or above. However we are prepared to evaluate the synthesis of MHC-peptide complexes that yield a score of 20 or lower if other conclusive binding data are available. If scoring with SYFPEITHI is not available for your allele of choice please contact us.

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