Research about dilution of SIINFEKL peptide 3-12-12
2015-01-13azim58 - Research about dilution of SIINFEKL peptide 3-12-12
- Here is info on the peptide I received: SINFEKL peptide 2-28-12 (Note
- Here's the link to the peptide on the SIGMA website:
o 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
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.
@ 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.
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
- 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
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/k2818u436069
3k26/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%2FS000887490300
128X&ei=HLBeT9DyK4i0sQLHirmkCA&usg=AFQjCNG2KWc5OZl9-laGq3dh_e
TG-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=PA
535&dq=what+should+the+SIINFEKL+peptide+be+dissolved+in&sourc
e=bl&ots=70bidisolu&sig=K1P55q35YTeDa1A9obKlflwc5wI&hl=en
&sa=X&ei=HLBeT9DyK4i0sQLHirmkCA&ved=0CGQQ6AEwBQv=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.
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I e-mailed SIGMA about this issue directly so hopefully their response
will be enlightening.
Response from Sigma about SIINFEKL dilution 3-14-12