Thursday, July 31, 2008

punkscience made a boo-boo


That HPLC method I've been working on- the one where I kept destroying columns, yeah? Well I think I've finally managed to work out how to avoid that little problem. However I've spent this week running standards through 'Polly' (for so she is named) and failing to find the nice peak I should be getting. Hrrrrrrmmmmmmm . . . . . ?

I thought I had it the other week before I killed my previous column, however I couldn't repeat it with my new column that I "acquired" from a friendly lecturer at my old Uni. This was particularly vexing and I was up until 0230 on Wednesday morning and 0330 this morning running samples and changing gradients and generally tinkering in an attempt to find my peak of interest. I went to bed last night decidedly downhearted, having failed to do so. So this morning was a miserable experience what with the lack of data, the ticking clock and the fact that I had offered to use this technique- which I couldn't make work- to do some analysis for a student of the aforementioned friendly lecturer as a gesture of thanks. Hence, driven by my usual tenacity when problem-solving, I found myself reading through several of the methods which lead up to the development of the one I am applying. Reading through the derivatisation technique I was struck by a sudden epiphany: I had been using the wrong buffer!

The assay for total and oxidised glutathione requires different preparations for each fraction, the former requiring preparation in simple PBS and the latter in a solution of N-ethylmaleimide to covalently bond to any reduced glutathione present and block it from subsequent detection. The oxidised glutathione is then chemically reduced and the assay continues as with that for the reduced fraction, which needs no special blocking. After the relevant blocking & reduction steps reduced glutathione is derivatised with a thiol-specific fluorescent probe called bromobimane in a buffer of N-ethylmorpholine. This latter reagent sounds rather similar to the thiol- masking one and in preparing my protocol I had inadvertently used the same acronym for both- "NEM"- and didn't notice that they were actually meant to be completely different reagents! The upshot of this simple mistake was two weeks spent adding a compound that specifically masks thiols to a reaction where those same thiols were meant to be fluorescently labelled for subsequent detection!

Having realised and corrected this mistake I ran some freshly prepped standards through Polly today and was overjoyed to produce this:

The peaks on the left of the image are a series of 5mM glutathione standards. The area under the peak is directly proportional to the concentration. Once again, I rule.

Ho hum. We are all human, it seems. Even someone as profoundly awesome as me. Buggering arsewank goatpants, is all I have to say. Now I can proceed with the cutting edge science and then move on to saving the world and becoming its God-Emperor.


1 comment:

  1. Hey you have some nice peaks there. Im currently trying to measure Cysteine/Cystine and Gluthathion in RP-HPLC after reduction with TCEP and derivatisation with Bromobimane, but the protocol im using only produces shitty non linear strange peaks. Could I have a look on the protocol that works for you?
    Thank you so much and best regards from Auckland!



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