FAQ

How do I request reagents?
When do I find out if my requests get approved?
Where is my confirmation email?
When do I receive my reagents?
How much do reagents cost?
What if the MHC allele I want isn't listed as available?
Do I need to send anything?
Do you ship internationally?
Do I need to have an NIH grant?
Do you support corporate clients?
How do I make tetramers from monomers?
Why Human b2m?
Shelf life and storage?


How do I request reagents?

A detailed summary of the request process can be found on the Request Process page, under the Reagents menu.

All clients are required to have a Materials Transfer Agreement between a Principal Investigator at their respective Institution and the NIH Tetramer Core Facility.  Click here to read more about the MTAs and to download the MTA form.


When do I find out if my requests get approved?

New requests are reviewed approximately every two weeks by the Tetramer Resource Committee (TRC) at the NIH.  You can see their meeting schedule here.  Any request received by 12PM Eastern Time the day before each TRC meeting will be reviewed at that respective meeting.  If your request is approved, you should receive an email within a few business days giving you Task Order numbers for your requests, and further instructions.

Sometimes, the TRC may require more information from you before your request can be approved.  In this case, you would also be contacted within a few business days of the review for this information.


Where is my confirmation email?

The confirmation email is generated and sent on its way to you by the time you see the "Thank you for your request" page.  It is rare for it to take as long as 15 minutes to be delivered, although some client organizations have email servers that can cause long delays in delivering emails.

The most common reasons for our clients to not receive their confirmation emails are that the email address is not entered correctly on your account (so it was not sent to the expected address), or else the client's "spam filter" did not permit our email to go through (check your Spam or Junk folder).  We receive inquiries about missing email for fewer than 1% of our system-generated emails.


When do I receive my reagents?

It depends on what you are requesting.

Custom Class I - If we have not made this reagent for you in the past, you must first send us epitope peptide with which to synthesize your reagent.  Average turnover time is 4-6 weeks from the receipt of peptide.  If we have made this request for you in the past, there is a chance we still have some available from the previous synthesis.  In cases like this the reagent may be sent within a week or two of receiving approval.

CD1 or Ligands - In most cases, these reagents can be shipped within a week or two of receiving approval.

Premade Class II - In most cases, these reagents can be shipped within a week or two of receiving approval.  There are occasions, however, when we run out of stock of a particular reagent and must produce more.  In this case it may take 3-6 weeks to receive your reagents.

Custom Class II - These reagents require construction of new cell lines, which is a time consuming process.  Availability is expected to be between 2 and 4 months after receiving approval.


How much do reagents cost?

There is no charge for the synthesis of reagents from this facility.  All costs, except shipping and the supplying of peptide in cases where it is needed, are borne by the U.S. National Institutes of Health.  


<p>
<a name="top"></a><strong>FAQ</strong><br />
<br />
<a href="#howrequest">How do I request reagents?</a><br />
<a href="#when">When</a><a href="#when">&nbsp;do I find out if my requests get approved?</a><br />
<a href="http://yer02cw1p-vm04.emory.edu/tetramer_new/node/27/edit#cost">How much do reagents cost?</a><br />
<a href="#outside">Do you ship internationally?</a><br />
<a href="#1">How do I make tetramers from monomers?</a><br />
<a href="#2">Why Human b2m?</a><br />
<a href="#3">Shelf life and storage?</a></p>
<hr />
<p>
<strong><a name="howrequest"></a>How do I request reagents?</strong><br />
All requests, whether new or for refills, should be placed through our online request form. &nbsp;(http://tetramer.yerkes.emory.edu/request/request)</p>
<p>
All clients are required to have a Materials Transfer Agreement between a Principal Invesigator at their respective Institution and the NIH Tetramer Core Facility. &nbsp;A link to our form can be found here. (http://tetramer.yerkes.emory.edu/request/material)</p>
<hr />
<p>
<strong><a name="when"></a>when&nbsp;do I find out if my requests get approved?</strong><br />
New requests are reviewed approximately every two weeks by the Tetramer Resource Committee (TRC) at the NIH. &nbsp;You can see their meeting schedule here (http://tetramer.yerkes.emory.edu/request/schedule). &nbsp;Any request received by <strong>12PM EST the day before each TRC meeting</strong> will be reviewed at that respective meeting. &nbsp;If your request is approved, you should receive an email within a few business days giving you Task Order numbers for your orders and further instructions.</p>
<p>
Sometimes, the TRC may require more information from you before your request can be approved. &nbsp;In this case, you would also be contacted within a few business days of the review for this information.</p>
<meta charset="utf-8" />
<hr />
<p>
<strong><a name="cost"></a>How much do reagents cost?</strong><br />
There is&nbsp;<strong>no charge</strong>&nbsp;for reagents from this facility.&nbsp; All costs, except shipping, are borne by the U.S. National Institutes of Health.&nbsp; Generally, the NIH approves requests for tetramers to advance scientific research done by non-profit entities.&nbsp; If you are looking for tetramers for a commercial (for-profit) purpose, it&nbsp;<em>may</em>&nbsp;be possible to get approval.</p>
<hr />
<p>
<strong><a name="outside"></a>Do you ship internationally?</strong><br />
Yes.&nbsp; We have many clients outside the U.S., on every populated continent.</p>
<hr />
<p>
<a name="1"></a><strong>How do I make tetramers from monomers?</strong><br />
It is very easy to prepare tetramers from monomers. You add the appropriate amount of streptavidin-conjugate to your biotinylated monomer in ten portions, adding one aliquot every ten minutes.</p>
<p>
You can purchase Streptavidin-PE and Streptavidin-APC from Molecular Probes (Cat # S-866 and S-868, respectively). The total volume of streptavidin-conjugate to add will vary based on the molecular weight of the monomer, the % biotinylation, as well as the molecular weight of the streptavidin conjugate.</p>
<p>
<strong>Materials Needed</strong><br />
Your Favorite Fluorescent Label conjugated to Streptavidin<br />
Pipet</p>
<p>
<strong>Background</strong><br />
MHC tetramers are typically prepared with the fluorescent labels most commonly used for flow cytometry. The choice of label is based upon the signal-to-noise ratio of the label, the capabilities of the flow cytometer that will be used for the assay, and the availability of other fluorescent antibodies to be used in conjunction with the tetramer. Tetramers labeled with phycobiliproteins such as phycoerythrin (PE) or allophycocyanin (APC) usually give the brightest signals and have been used in most experiments. In our lab, we prefer to use the APC label for the tetramers, reserving the FITC or PE channels for other commercially available phenotyping reagents. However, many commonly used flow cytometers do not have the capacity to measure APC (e.g. the FACScan, the Coulter EPICS XL); when using these instruments, PE is the recommended choice for the tetramer label.</p>
<p>
<strong>Protocol</strong></p>
<ol>
<li>
<strong>Calculate the total moles of biotinylated monomer in the purified MHC stock.</strong><br />
The NIH Tetramer Facility typically provides biotinylated monomers in 100 ul aliquots at a concentration of 2 mg/ml, and the total nmole of biotinylated monomer (calculated from the molecular weight of the monomer and the percent biotinylation) is listed on your packing slip.</li>
<li>
<strong>Calculate the concentration of biotin binding sites in the streptavidin-fluorophore conjugate stock.</strong><br />
To perform this calculation, it is necessary to know the concentration of the streptavidin conjugate, whether the concentration is expressed in terms of streptavidin content alone or in terms of the total mass of streptavidin conjugate, and the ratio of label to streptavidin. As an example, Molecular Probes provides streptavidin-PE conjugates at a concentration of 1 mg/ml. The concentration refers to the mass of the total conjugate, not just the streptavidin moiety. In addition, the material from Molecular Probes is size selected to have a 1:1 ratio of phycoerythrin:streptavidin. The average molecular weight of a 1:1 streptavidin-PE conjugate is 300,000 Da. Therefore, the concentration of biotin binding sites is:<br />
<br />
(1 mg SA-PE/mL) * (1 g/1000 mg) * (1 mL/ 1000 uL)* (1 mol SA-PE/300,000 g) * 4 biotin binding sites/mol SA-PE) = 1.33 x 10-11 mol biotin binding sites per uL.<br />
<br />
The table below gives the average number of biotin binding sites for three common streptavidin conjugates.
<table border="0" cellpadding="2" cellspacing="0" width="100%">
<tbody>
<tr valign="top">
<td width="25%">
<strong>Label</strong></td>
<td width="25%">
<strong>Molecular Weight</strong></td>
<td width="25%">
<strong>Concentration,<br />
mg/ml</strong></td>
<td width="25%">
<strong>moles biotin binding<br />
sites per uL</strong></td>
</tr>
<tr class="even">
<td>
Phycoerythrin</td>
<td>
300,000</td>
<td>
1</td>
<td>
1.33 x 10-11</td>
</tr>
<tr>
<td>
Allophycocyanin</td>
<td>
164,000</td>
<td>
1</td>
<td>
2.44 x 10-11</td>
</tr>
<tr class="even">
<td>
FITC</td>
<td>
60,000</td>
<td>
1</td>
<td>
6.67 x 10-11</td>
</tr>
</tbody>
</table>
</li>
<li>
<strong>Calculate the volume of streptavidin-fluorophore conjugate that must be added to give a 1:1 ratio of biotinylated monomer to biotin binding sites.</strong><br />
For example, when adding streptavidin-APC to 200 ug of H-2D(b)/ ASNENMETM (MW 47275, 95% biotinylation) , the proper total volume of streptavidin-APC is 165 ul.</li>
<li>
<strong>Add the streptavidin slowly, in 1/10 volume aliquots, waiting 10-15 minutes between each addition.</strong><br />
This process is performed at room temperature, and the tube containing the MHC and streptavidin is kept in the dark (e.g., in a drawer or under foil). Some researchers will do this on ice because some monomers can be unstable at room temperature (although this is rare).<br />
<br />
If the volume of streptavidin required has been overestimated, this procedure will produce the maximum concentration of tetramers because all biotin binding sites will be saturated after the early additions.</li>
<li>
<strong>Store the tetramer at 4&infin;C in an amber polypropylene tube in a covered box. Tetramer stocks prepared in this way have been used for 3-12 months.</strong><br />
As with most phycobiliprotein conjugates, freezing of the tetramers is not generally recommended, although reagents that have been frozen may retain some activity. Although we have not investigated this, storage of tetramers in the presence of excess peptide and/or beta-2m may increase the stability of the reagents.</li>
</ol>
<p>
<a href="#top">top</a></p>
<hr />
<p>
<strong><a name="2"></a>Why does the Tetramer Core Facility use human B2m when making reagents with mouse alleles?</strong><br />
Human b2m has been shown to bind more tightly to mouse class I alleles than mouse b2m.<br />
<cite>(Pedersen LO, Stryhn A, Holter TL, Etzerodt M, Gerwien J, Nissen MH, Thogersen HC, and Buus S. 1995. The interaction of beta 2-microglobulin (beta 2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding: A comparison of human and mouse beta 2m. Eur J Immunol. 25(6): 1609-16).</cite><br />
Though we have not tested this systematically, we reasoned that the higher affinity binding of human b2m ought to lead to higher refolding yields. In addition, there is plenty of evidence that reagents with human b2m work well for identification of antigen-specific T cells, and no evidence that reagents with mouse b2m are superior for conventional applications of MHC tetramers.</p>
<p>
<strong>Under what circumstances is mouse b2m required in mouse class I tetramers?</strong><br />
It has been shown by x-ray crystallography and biochemical binding data that the natural killer cell inhibitory receptor Ly49A makes crucial contacts with residues in murine b2m in H-2D(d) that are mutated in human b2m, leading to reduced binding.<br />
<cite>(Wang J, Whitman MC, Natarajan K, Tormo J, Mariuzza RA, and Margulies DH. 2002. Binding of the natural killer cell inhibitory receptor Ly49A to its major histocompatibility complex class I ligand. Crucial contacts include both H-2Dd and beta 2-microglobulin. J Biol Chem. 277(2): 1433-42).</cite><br />
In addition, Ly49C has also been shown to contact b2m residues in H-2Kb complexes, though the binding mode appears to be different and it is not clear if these residues are mutated in human b2m.<br />
<cite>(Dam J, Guan R, Natarajan K, Dimasi N, Chlewicki LK, Kranz DM, Schuck P, Margulies DH, and Mariuzza RA. 2003. Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b). Nat Immunol. 4(12): 1213-22).</cite><br />
Reagents with mouse b2m may also be required if you are conducting certain in vivo studies and want to avoid generation of mouse-anti-human b2m antibodies.</p>
<p>
<a href="#top">top</a></p>
<hr />
<p>
<strong><a name="3"></a>How long will the tetramer last?</strong><br />
There is not one specific answer to that question. The stability and shelf life of our reagents depends on the particular allele/peptide combination. Typically tetramer stocks stored at 4&infin;C protected from the light are stable for 3-6 months, although some researchers have reported using our tetramers for one year or more. The best source of information on this topic is our clients. We request that all clients fill out our <a href="../client/feedback">online feedback forms</a> to help us compile data on the shelf life of our reagents.</p>
<p>
<strong>How should the tetramer be stored?</strong><br />
We recommend that MHC class I and class II tetramers be stored at 4&infin;C. While some investigators report storing tetramers at -20&deg;C or -80&deg;C, we have not tested the effect of freezing on tetramers. Also the manufacturer of the streptavidin-PE and streptavidin-APC solutions we use cautions against freezing.</p>
<p>
We recommend that biotinylated monomers not yet labeled with a streptavidin-fluorophore conjugate be stored at -80&deg;C.</p>
<p>
<strong>How is the tetramer packaged?</strong><br />
We prepare biotinylated monomer stocks in phosphate-buffered saline containing 1 ug/mL Leupeptin, 1 uM Pepstatin, and 2 mM EDTA, typically at a concentration of 2 mg/mL.</p>
<p>
This stock is diluted when the streptavidin-fluorophore conjugate is added to tetramerize the reagent. PE-, APC-, and small molecule-labelled tetramers are diluted out by a factor of 4, 2.7, and 1.7, respectively.</p>
<p>
The exact volume and concentration of monomer will be listed on the packing slip that comes with your reagent, as well as the volume of streptavidin-fluorophore conjugated added.</p>
<p>
The SA- PE and SA-APC solutions used to make tetramers contain 2 mM sodium azide that is diluted out to ~1.5 or 1.25 mM in the final tetramer solution.</p>
<p>
<strong>What is the concentration of tetramer in the tube we receive from the NIH Tetramer Facility?</strong><br />
The final concentration of tetramer varies depending on the fluorescent label used. For most tetramers the approximate concentrations are</p>
<p>
~10uM for PE-labeled tetramers<br />
~15uM for APC-labeled tetramers<br />
~25uM for Fluorescein-labeled tetramers<br />
~25uM for Alexa 488-labeled tetramers<br />
~25uM for Alexa 647-labeled tetramers<br />
~25uM for Alex 680-labeled tetramers<br />
~25uM for Pacific Blue-labeled tetramers.</p>
<ul>
</ul>
<p>
For more exact values, please consult the packing slip you received with your reagent.</p>
<p>
<a href="#top">top</a></Do I need to send anything?

What if the MHC allele I want isn't listed as available?

Please see the Unlisted Reagents page.


Do I need to send anything?

It depends on what you are requesting. We will send you a specific list of what you need to send when your requests are approved.

If you are ordering a CD1, ligand, or premade class II reagent, you don't need to send anything.

If you are ordering a custom class I reagent, we may need to receive peptide from you in order to conduct the synthesis.  If you are ordering a refill, we may have more peptide on hand from your previous shipment.

If you are ordering a custom class II reagent, you often do not need to send anything.  However, there are a few cases, particularly with Human class II, where we may attempt to produce your reagent by a process called peptide exchange.  In this case, you would need to send us a small amount of peptide as well.

In any case where you are requesting an allele that we haven't worked with before, we might ask you to send cDNA, however we make many new alleles by ordering synthetic genes.

If you need to send peptide or cDNA, these items can be sent to:

Dale Long
NIH Tetramer Facility at Emory/Yerkes
954 Gatewood Road
Atlanta, GA  30329


Do you ship internationally?

Yes.  We have many clients outside the U.S., on every populated continent.  Some countries have more difficulties with Customs than others, so make sure you have any documents your country may require ready to provide.



Do I need to have an NIH grant?

No, an NIH grant is not required to use our services.


Do you support corporate clients?

Generally, the NIH approves requests for tetramers to advance scientific research done by non-profit entities.  If you are looking for tetramers for a commercial (for-profit) purpose, it may be possible to get approval.  Permission must be granted by Beckman-Coulter Immunomics (BCI), who holds special licensing agreements on the technology.  Contact Dr. Alison Deckhut-Augustine for more information.


How do I make tetramers from monomers?

It is very easy to prepare tetramers from monomers, please see Production Protocols.


Why does the Tetramer Core Facility use human β2m when making reagents with mouse alleles?

Human β2m has been shown to bind more tightly to mouse class I alleles than mouse b2m.
(Pedersen LO, Stryhn A, Holter TL, Etzerodt M, Gerwien J, Nissen MH, Thogersen HC, and Buus S. 1995. The interaction of beta 2-microglobulin (beta 2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding: A comparison of human and mouse beta 2m. Eur J Immunol. 25(6): 1609-16).
Though we have not tested this systematically, we reasoned that the higher affinity binding of human b2m ought to lead to higher refolding yields. In addition, there is plenty of evidence that reagents with human β2m work well for identification of antigen-specific T cells, and no evidence that reagents with mouse b2m are superior for conventional applications of MHC tetramers.


Under what circumstances is mouse β2m required in mouse class I tetramers?
It has been shown by x-ray crystallography and biochemical binding data that the natural killer cell inhibitory receptor Ly49A makes crucial contacts with residues in murine β2m in H-2D(d) that are mutated in human β2m, leading to reduced binding.
(Wang J, Whitman MC, Natarajan K, Tormo J, Mariuzza RA, and Margulies DH. 2002. Binding of the natural killer cell inhibitory receptor Ly49A to its major histocompatibility complex class I ligand. Crucial contacts include both H-2Dd and beta 2-microglobulin. J Biol Chem. 277(2): 1433-42).
In addition, Ly49C has also been shown to contact b2m residues in H-2Kb complexes, though the binding mode appears to be different and it is not clear if these residues are mutated in human b2m.
(Dam J, Guan R, Natarajan K, Dimasi N, Chlewicki LK, Kranz DM, Schuck P, Margulies DH, and Mariuzza RA. 2003. Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b). Nat Immunol. 4(12): 1213-22).
Reagents with mouse β2m may also be required if you are conducting certain in vivo studies and want to avoid generation of mouse-anti-human b2m antibodies.


How long will the tetramer last?
There is not one specific answer to that question. The stability and shelf life of our reagents depends on the particular allele/peptide combination. Typically tetramer stocks stored at 4°C protected from the light are stable for 3-6 months, although some researchers have reported using our tetramers for one year or more. The best source of information on this topic is our clients. We request that all clients fill out our online feedback forms to help us compile data on the shelf life of our reagents; these can be found in your user menu at the top of the page.


How should the tetramer be stored?
We recommend that MHC class I and class II tetramers be stored at 4°C. It is generally recommended that PE and APC not be frozen.

The TCF has tested class I tetramers with multiple labels in freeze-thaw experiments, and found that PE, APC and Alexa dye labeled tetramers can be frozen at -20°C or -80°C by adding glycerol to a final concentration of 20%. Please note that this was one freeze-thaw cycle, and we cannot be sure all tetramers will still stain after freezing using this method.

We recommend that biotinylated monomers not yet labeled with a streptavidin-fluorophore conjugate be stored at -80°C.


How is the tetramer packaged?
We prepare biotinylated monomer stocks in phosphate-buffered saline containing 1 ug/mL Leupeptin, 1 uM Pepstatin, and 2 mM EDTA, typically at a concentration of 2 mg/mL.

This stock is diluted when the streptavidin-fluorophore conjugate is added to tetramerize the reagent. PE-, APC-, and small molecule-labelled tetramers are diluted out by a factor of 4, 2.7, and 1.7, respectively.

The exact volume and concentration of monomer will be listed on the packing slip that comes with your reagent, as well as the volume of streptavidin-fluorophore conjugated added.

The SA- PE and SA-APC solutions used to make tetramers contain 2 mM sodium azide that is diluted out to ~1.5 or 1.25 mM in the final tetramer solution.


What is the concentration of tetramer in the tube we receive from the NIH Tetramer Facility?
The final concentration of tetramer varies depending on the fluorescent label used. For most tetramers the approximate concentrations (in terms of MHC) are

~10uM for PE-labeled tetramers
~15uM for APC-labeled tetramers
~25uM for Fluorescein-labeled tetramers
~25uM for Alexa 488-labeled tetramers
~25uM for Alexa 647-labeled tetramers
~25uM for Alexa 680-labeled tetramers

    For more exact values, please consult the packing slip you received with your reagent.

    For any other technical inquiries not addressed above, you may contact our Technical Director, Rick Willis, at richard.willis@emory.edu