Quantification of peptide Total GIP - (Feb/23/2011 )

Hi All,
I am working on Total GIP(1-42).

I am having some difficulties with the peptide. I have an ELISA kit from Millipore company, and I have bought GIP peptide from a different company. The company has provided me the peptide in lyophilized form and I reconstituted it in water to make a concentration of 100ug/ml and then froze the aliquots. Now when I am running this peptide in the ELISA kit against my standard curve, it does not give me the expected concentration. The concentration is around 10 fold less and it varies when I repeat the experiment also the dilutions are not linear.

Can any of this behavior explained by the peptides structure, or it being hydrophobic or any other thing you can think of. I have tried talking to Millipore company but they do not have an insight about it.

Best wishes,

Anisha

just a couple of questions.

are you sure that the peptide was completely solubilized?

was it mixed after defrosting?

are your pipettes calibrated?

are you sure that the peptide did not bind to the tube?

was there salt and/or buffer in the lyophilized peptide? if not then you may need to add some to prevent nonspecific binding to the tube, tip, etc.

I think the peptide was completely solublized because I called the vendor I bought it from they asked me to dissolve it in water.

I vortexed it after thawing and right before I used it.

Our pipettes are calibrated.

How can I check if the peptide was bind to the tube?

there was salt in the peptide.

Hi performed a BCA test on the peptide it is still not linear.

Anisha Kharkia on Thu Feb 24 18:45:33 2011 said:

do you mean not on the linear portion of the standard curve or 2x volume does not give you 2x the response?

does the data sheet say that you have 100ug peptide or 100ug solid? the weight may include buffer salts.

salt helps prevent nonspecific binding to the tube

I mean 2x of the volume does not give 2x of the response.

Also yes I took that into account, and after removing the salt , the concentration should be 100 ug/ml.

I called up the company I bought peptide from and they think I am diluting it in a buffer which has high pH of 8.5, they make there peptide using TFA which has buffer pH=5,
I dont have any TFA. Can you suggest any other buffer I can make in lab, which will have pH=5.

I am thinking of using TRIS-HCL ph 7.5 which I have and adding HCL in it to make it pH=5. Do you think HCL will effect peptide in any sort? Is this a good approach ? Any suggestions.

Thanks,
Anisha

don't use tris for pH 5. it is not a buffer at that pH.

you can use acetate, citrate, malate, succinate, maleate, benzoate, phthalate... any buffer with a pK within 1 pH unit of your desired pH.

you can extend the buffering range by blending with other (overlapping) buffers.