Research+about+dilution+of+SIINFEKL+peptide+3-12-12

>>>> purified OVA257-64 (SIINFEKL) peptide was purchased from Anaspec (San >>>> Jose, CA). MHC-I-DimerX:mouse-Ig:OVA257-64 conjugates were prepared >>>> according to manufacturer’s instructions (BD Biosciences). Peripheral >>>> blood was incubated with DimerX:mouse-Ig:OVA257-64-PE plus antibodies >>>> for other markers (all from BD Biosciences), and 5  103 donor CD8 T >>>> cells were collected and analyzed by flow cytometry. >>>> >>>> 29. Stotz, S. H., Bolliger, L., Carbone, F. R. & Palmer, E. T cell receptor (TCR) antagonism without a >>>> negative signal: evidence from T cell hybridomas expressing two independent TCRs. J. Exp. Med. 189, >>>> 253–264 (1999). >>>> >>>> Peptides. The peptides were synthesized at the Basel Institutefor Immunology using FastMocTM chemistry on 430A peptidesynthesizer (Applied Biosystems). The amino acid sequences werethe following: SIINFEKL (Va2Vb5-specific antigen), EIINFEKL(Va2Vb5-specific antagonist E1), SIINFEPL (Va2Vb5-specificantagonist P7), SIIKFEKL (the control peptide K4), and SSIEFARL(Va2Vb10-specific antigen). >>>> This didn't tell me anything about how to dilute the peptides >>> what should the SIINFEKL peptide be dissolved in >>> >>> http://www.anaspec.com/html/peptide_tips.html >>> Peptides are usually supplied as a fluffy, freeze-dried material in serum vials. Store peptides in a freezer after they have been received. In order to reconstitute the peptide, distilled water or a buffer solution should be utilized. Some peptides have low solubility in water and must be dissolved in other solvents such as 10% acetic acid for a positively charged peptide or 10% ammonium bicarbonate solution for a negatively charged peptide. Other solvents that can be used for dissolving peptides are acetonitrile, DMSO, DMF, or isopropanol. Use the minimal amount of these non-aqueous solvents and add water or buffer to make up the desired volume. After peptides are reconstituted, they should be used as soon as possible to avoid degradation in solution. Unused peptide should be aliquoted into single-use portions, relyophilized if possible, and stored at -20°C. Repeated thawing and refreezing should be avoided. >>> >>> For a very hydrophobic peptide, try dissolving the peptide in a very small amount of DMSO, dilute with water to the desired concentration. >>> >>> Is SIINFEKL a hydrophobic peptide? >>> Yes it does appear to be fairly hydrophobic. >>> tool used for determination: http://haubergs.com/peptide >>> 4 hydrobphobic amino acids with a total hydropathy score of 1.56 >>> >>> It looks like there are several research papers in which they dissolve the SIINFEKL peptide in H20 or PBS. >>> >>> This one actually uses DMSO >>> http://www.jimmunol.org.ezproxy1.lib.asu.edu/content/184/10/5663.full.pdf+html >>> OVA257–264 peptide, a H-2Kb–restricted epitope with amino acid sequence >>> SIINFEKL, was purchased from Auspep (Melbourne, Australia) with .80% >>> purity, dissolved in 100% DMSO and stored at 220°C. >>> >>> Tracing Tolerance and Immunity In Vivo by CFSE-Labeling of Administered Cells >>> not accessible >>> http://www.springerlink.com.ezproxy1.lib.asu.edu/content/k2818u4360693k26/#section=85137&page=3&locus=24 >>> OVA257-264 synthetic peptide (SIINFEKL; Research Genetics) dissolved in PBS at 200-mM stock and stored at -80 C in small aliquots for addition to cell suspensions as required >>> >>> >>> Peptide-loaded dendritic cells prime and activate MHC-class I-restricted T cells more efficiently than protein-loaded cross-presenting DC >>> http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=5&cts=1331608595333&ved=0CF8QFjAE&url=http%3A%2F%2Fwww.sciencedirect.com%2Fscience%2Farticle%2Fpii%2FS000887490300128X&ei=HLBeT9DyK4i0sQLHirmkCA&usg=AFQjCNG2KWc5OZl9-laGq3dh_eTG-PGGeg&sig2=f1fZtYLgQsllK21IWv68_g >>> 2.4. Peptides and protein >>> H-2Kb binding OVA257–264-peptide (SIINFEKL; MW=963 Da) and I-Ab binding OVA323–339-peptide (ISQAVHAAHAEINAGR; MW=1772 Da) were synthesized by Shafer-N (Copenhagen, Denmark). Peptides were dissolved in distilled water, freeze-dried, and stored at -20 °C. OVA (Sigma, Steinheim, Germany, MW=44.287 Da) was dissolved in PBS and stored at 4 °C. >>> >>> >>> Applications of chimeric genes and hybrid proteins: Gene ..., Volume 326 >>> edited by Jeremy Thorner, Scott Emr, John Abelson >>> http://books.google.com/books?id=Ff1V02witwoC&pg=PA535&lpg=PA535&dq=what+should+the+SIINFEKL+peptide+be+dissolved+in&source=bl&ots=70bidisolu&sig=K1P55q35YTeDa1A9obKlflwc5wI&hl=en&sa=X&ei=HLBeT9DyK4i0sQLHirmkCA&ved=0CGQQ6AEwBQ#v=onepage&q=what%20should%20the%20SIINFEKL%20peptide%20be%20dissolved%20in&f=false >>> The B3Z hybridoma was also stimulated strongly by Thio-PEC in the presence of the synthetic OVA peptide [SIINFEKL, synthesized by Neosystem, Strasbourg, France, and purified by reversed-phase high-performance liquid chromatography (HPLC); stock solutions of peptide were prepared in phosphate-buffered saline (PBS) and stored at -20], but not in the presence of ovalbumin, as expected >>> >>> GenScript has some good information about the peptide in general >>> They also have the following statement >>> The peptide was water soluble on vortexing. May be dissolved in any buffer of pH <6. >>> -I hope they actually meant pH > 6. A lot of the other papers just use H20 or PBS which has a pH of about 7.5 >>> >>> Here's some information from a patent >>> http://www.patentstorm.us/patents/6210672/description.html >>> C57BL/6 mice were treated as follows: 1) shaving alone; 2) topical administration of acetone and dibutylphthalate; 3) SIINFEKL (SEQ ID NO: 1) (240 µg/ml) in DMSO diluted in PBS, followed in 5 hours by acetone and dibutylphthalate; and >>> ALSO >>> Lipophilic solvents like acetone, azone, and dimethylsulfoxide (DMSO) are known to penetrate the stratum corneum. As discussed above, the inventor has found that small peptides, like SIINFEKL, dissolved in DMSO cross the stratum corneum and enter the lower strata of the epidermis. Thus, the present disclosure with respect to antigen penetration enhancers, encompasses a variety of lipophilic solvents, including DMSO, and symmetrical or unsymmetrical sulfide and sulfoxides where the alkyl group contains 1 to 16 carbon atoms, as well as liposomes. >>> Where lipophilic penetrants, such as DMSO, are employed in the present invention to enhance peptide penetration, such solvents may be administered neat or diluted with other solutions, such as PBS. Ratios of mixtures may range from about 1:9 to about 9:1 (penetrant to diluant). Preferably, the penetration enhancer is undiluted. >>> >>> paper about concentration required for killing >>> http://jem.rupress.org/content/207/1/223.full.pdf >>> We used B16 cells pulsed with 10?6 M SIINFEKL peptide inall subsequent experiments.
 * Here is info on the peptide I received: SINFEKL peptide 2-28-12 (Note that the text should have been SIINFEKL (with two Is))
 * Here's the link to the peptide on the SIGMA website: http://www.sigmaaldrich.com/catalog/product/sigma/s7951?lang=en®ion=US
 * they have three papers as references. These papers might indicate the best thing to dissolve the peptide in.
 * Kelly RM, et al., Keratinocyte growth factor and androgen blockade work in concert to protect against conditioning regimen-induced thymic epithelial damage and enhance T-cell reconstitution after murine bone marrow transplantation. Blood 12th ed., 111, 5734-5744, (2008)
 * MHC-I-DimerX:mouse-Ig-PE was purchased from BD Biosciences, and
 * Nothing about how to dilute the peptide
 * Werlen G, et al., A motif in the alphabeta T-cell receptor controls positive selection by modulating ERK activity. Nature 6794th ed., 406, 422-426, (2000)
 * SIINFEKL and EIINFEKL peptides were synthesized as described29.
 * Hogquist KA, et al., T cell receptor antagonist peptides induce positive selection. Cell 1st ed., 76, 17-27, (1994)
 * a quick search didn't reveal any of the information I was after
 * Google search

I e-mailed SIGMA about this issue directly so hopefully their response will be enlightening. Response from Sigma about SIINFEKL dilution 3-14-12