We are dealing with a silver stained gel band that is thought contain a
receptor &/or a metalloprotein molecule. We experienced a strange series
of color changes in processing that we have not encountered before. We are
concerned that the final site of the color indicated the location of the
analyte of interest, since it was NOT in the supernatant post in-gel
tyrptic digestion as determined by LC/ tandem MS. The following is what
happened:
First the excised silver stained gel band was destained using Farmers
reagent (Na2S2O3 & K3Fe(CN)6). The black/brown color disappeared and the
gel piece turned blue.
On reducing with DTT, the blue color disappeared and a light pink color
tinted the gel band. Please note that it was PINK not pale yellow or pale
red.
When alkylating with Iodoacetamide, the light pink color turned to light
blue.
Next, an in-gel digestion with trypsin using the modified Hellman protocol
was performed. The band was extracted with 50% ACN/ 5% Formic acid. The
blue color remained in the gel residue. The LC/MS/MS showed only strong
Keratin features.
We have seen Keratin before in Silver stained samples but never this color
change. Do you think that some PAH tagging along is responsible for the
color changes? Is it likely that Keratin is all that is present in this
gel band or is our analyte being masked? Do metalloproteins behave this
way?
Please advise. This is a precious sample that the client has been trying
to isolate for 2 years.
Thank you!!
Mary
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