Product Information

Contents: Affinity Purified anti-mouse MHC class I molecule Kb bound to the peptide SIINFEKL (Kb-SIINFEKL) H-2Kb, OVA agonist peptide (OVAp)
Catalog Number: 14-5743
Sizes: 50 ug, 100 ug
Formulation: Phosphate buffered saline, pH 7.2, less than or equal to 0.09% NaN₃
Storage Conditions: Store at 4°C.
Avoid repeated freeze/thaw cycles.
Clone: eBio25-D1.16 (25-D1.16)
Isotype: Mouse IgG1, κ

Staining of C57Bl/6 splenocytes, either unpulsed (left) or pulsed with the SIINFEKL peptide (right), with 0.125 μg of Purified Mouse IgG1, K Iso Cntrl (cat. 14-4714) (open histogram) or 0.125 μg of Purified anti-mouse MHC class I Kb-SIINFEKL (25-D1.16) (colored histogram) followed by PE Donkey F(ab')2 Fragment Anti-Mouse IgG (H+L)(Minimal Reactivity to Rat IgG) (cat. 12-4012). Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
14-5743	Affinity Purified anti-mouse MHC class I molecule Kb bound to the peptide SIINFEKL (Kb-SIINFEKL) H-2Kb, OVA agonist peptide (OVAp)	N/A	N/A	FC IHC
13-5743	Biotin anti-mouse MHC class I molecule Kb bound to the peptide SIINFEKL (Kb-SIINFEKL) H-2Kb, OVA agonist peptide (OVAp)	N/A	N/A	FC IHC
12-5743	Phycoerythrin (PE) anti-mouse MHC class I molecule Kb bound to the peptide SIINFEKL (Kb-SIINFEKL) H-2Kb, OVA agonist peptide (OVAp)	488	575	FC
17-5743	Allophycocyanin (APC) anti-mouse MHC class I molecule Kb bound to the peptide SIINFEKL (Kb-SIINFEKL) H-2Kb, OVA agonist peptide (OVAp)	633	660	FC
Flow Cytometry Product Notes: Test Sizes: To accommodate multicolor flow cytometry, eBioscience is in the process of reducing test size volumes from 20 µl to 5 µl. Please check your antibody vial for the recommended test size. Fluorochrome Replacements: eBioscience is in the process of replacing all Pacific Blue® and APC-Alexa Fluor® 750 conjugated products with eFluor™ 450 and APC-eFluor™ 780 conjugated products, respectively.

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Description

The 25-D1.16 monoclonal antibody reacts with the ovalbumin-derived peptide SIINFEKL bound to H-2Kb of MHC class I, but not with unbound H-2Kb, or H-2Kb bound with an irrelevant peptide. This antibody has proven to be very useful tracking the quantity and localization of these specific antigen-presenting cells (APC) in vivo.

Applications Reported

For research use only, not for diagnostic or therapeutic use. This eBio25-D1.16 (25-D1.16) antibody has been reported for use in flow cytometric analysis, and immunohistochemical staining.

Applications Tested

This eBio25-D1.16 (25-D1.16) antibody has been tested by flow cytometric analysis of SIINFEKL-pulsed C57BL/6 splenocytes. This can be used at less than or equal to 0.25 μg per million cells in a 100 μl total staining volume. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.

Cells can be pulsed with the SIINFEKL peptide according to the following protocol:

1. With cells in flow staining buffer, add SIINFEKL peptide to a final concentration of 30 μM.

2. Incubate cells at 37°F for 2 hours.

3. Wash cells with flow staining buffer.

4. Proceed with cell surface staining as normal.

For additional information see the references listed below.

References

Porgador A, Yewdell JW, Deng Y, Bennink JR, Germain RN.Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody.Immunity. 1997 Jun;6(6):715-26. (25-D1.16, mAb development, PubMed)

Messaoudi I, LeMaoult J, Nikolic-Zugic J.The mode of ligand recognition by two peptide:MHC class I-specific monoclonal antibodies.J Immunol. 1999 Sep 15;163(6):3286-94.

Ackerman AL, Kyritsis C, Tampé R, Cresswell P.Access of soluble antigens to the endoplasmic reticulum can explain cross-presentation by dendritic cells.Nat Immunol. 2005 Jan;6(1):107-13.

Berwin B, Hart JP, Rice S, Gass C, Pizzo SV, Post SR, Nicchitta CV.Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells.EMBO J. 2003 Nov 17;22(22):6127-36.

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