SIGMA contact about impure SIINFEKL peptide 3-19-12

2015-01-13

azim58 - SIGMA contact about impure SIINFEKL peptide 3-19-12

I tried to perform an ELISPOT using splenocytes from mice immunized with
the ovalbumin protein and the SIINFEKL peptide which is a part of the
ovalbumin protein. The ELISPOT did not work. When we performed mass
spectrometry on the SIINFEKL peptide we ordered from SIGMA we found that
it was not very pure. You can see the mass spectrum here:
http://www.azim58.wikispaces.net/SIINFEKL+Peptide+MALDI+3-19-12

Do you think the impurity of this peptide may be the reason my ELISPOT
did not work? Is there some way that I can obtain a purer peptide from
SIGMA or perhaps obtain a refund or anything like that? Thanks for any
help and information you can offer me!

===========================================================================
Response

S7951, SIINFEKL trifluoroacetate salt

Sigma-Aldrich Technical Service Answer

Scientist's Response Via Email (Audrey Fleming)
 03/21/2012 09:58 AM

Hello Kurt,

Thank you for contacting Sigma-Aldrich Technical Service.

SIINFEKL is a class I (Kb)-restricted peptide epitope of ovalbumin (OVA);
the ovalbumin fragment is presented by the class I MHC molecule, H-2Kb. I
found literature using C57Bl6 mice for a similar experiment.

What are you using as the solvent for the Maldi? Is there any sodium
present? What is the pH? The difference between the 985.978 and the
expected 963.995 is 21.98 which would be equivalent to an exchange of a
sodium for a hydrogen. This can occur either on the glutamate or the
carboxy terminus of the peptide.

This product is tested for purity by HPLC and obtain a value of 97%. It
coelutes with the reference.
The peptide content is also indicated on the lot specific COA (attached)

I hope that you find this information helpful. If you have further
questions, please reply to this email.

Sincerely,
Audrey Fleming
Sigma-Aldrich Technical Service

Your Question By Email (Kurt Whittemore)
 03/20/2012 04:55 PM

Hi Audrey Fleming,

I have attached a document with the mass spectrometry data. This data is
hard to see, but can still be made out.

The lot number of the SIINFEKL peptide I used is 120M4708V.

I pulsed BALB/c CRL 2116 cells with the SIINFEKL peptide and incubated
with
splenocytes from Ova immunized BALB/c mice. I now see that this experiment
would not have worked even if the SIINFEKL peptide was very pure because
SIINFEKL is an H-2Kb restricted epitope and BALB/c cells have the H-2Kd
haplotype. Therefore, this was a major flaw in my experiment.
Nevertheless, the flaw in my experiment does not negate the fact that the
SIINFEKL peptide is certainly not very pure as you can see from the mass
spectra in the attached document.

Best regards,
Kurt Whittemore

On Tue, Mar 20, 2012 at 10:35 AM, Sigma-Aldrich Technical Service <
sigma_aldrich@mailnj.custhelp.com> wrote:

* **
>
>
>
>
>
>
>
>
>
>

* *>

========

application File Attachment ====================peptide_siinfekl.docx,
253984 bytes, added to incident

[---001:001799:45867---]

S7951 120M4708V COA.pdf
21K

===========================================================================

Kurt Whittemore <kurtwhittemore@gmail.com>
 Wed, Mar 21, 2012 at 9:09 AM

To: Sigma-Aldrich Technical Service <sigma_aldrich@mailnj.custhelp.com>

Ah okay, yeah it looks like you are correct. I diluted the peptide in
DMSO and PBS which does contain sodium. Therefore, I'm sure the peptide
actually is very pure.

I'll need to repeat my experiments and make sure that I have the correct
MHC haplotype for the peptide! Thanks for your help!

-Kurt

azim58wiki: