I would like to measure the concentration of T3 and T4 in zebrafish, and I'm trying to figure out the best way to extract the hormones from fish tissues. I tried extracting in phosphate buffer, but the concentrations were too low. Most of the protocols I find use barbital buffer, which I would like to avoid if at all possible, since it is a controlled substance and requires DEA certification. Any assistance would be greatly appreciated!
Hi Sarah,
the problem might be that most of the thyroid hormones are protein bound (serum albumin, receptors etc). So you'll need something that displaces the hormones from the proteins. Maybe high salt will do it, or adding chemically similar compounds which do not interfere with your assay.
Another thought was passing the tissue homogenate over immobilized antibodies, as they should have specific and probably stronger binding properties. Then elute the hormones with acidic buffer, then re-equilibrate to column to neutral for the next batch.
Edit: Get and compare the protocols for the determination of free and total thyroid hormones in serum from a department for lab diagnostics.
HTH
We've been extracting thyroid hormones from larval fish tissues for RIA analysis, as in the attached paper, but the extraction is based on the barbital buffer you're trying to avoid, sorry.
Article Thyroid and pituitary gland development from hatching throug...
Thanks, that protocol is helpful. Do you know why the barbital buffer is necessary? What does it do? Do you think it would work if I were to resuspend my samples in phosphate buffer? Thanks!
I also have been using barbital buffer to extract TH from Senegalese sole larvae. The barbital protocol has been described earlier by Tagawa & Hirano in 1987. I don't know the chemistry, but I have it seen described as that barbital facilitates solubilization of proteins and (hence?) the transfer of TH from the homogenate to (bovine serum) albumin, the final carrier for TH. I then use the barbital-BSA-TH extract in an ELISA for total (not free!) T3 or T4 measurement.
A colleague of mine, Joseph Schnitzler (Univ. Liege, Belgium) has used a barbital-free method to extract PCBs from sea bass muscle, and has used the same method to extract TH. See attachment and Schnitzler et al. 2011 Thyroid dysfunction in sea bass (Dicentrarchus labrax): Underlying mechanisms and effects of polychlorinated biphenyls on thyroid hormone physiology and metabolism. Aquat. Toxicol. 105, 438-47.
Also, I have this protocol that I never used but is in my notebook anyway. (I think Richard Manzon (Univ. of Regina), working on lamprey, sent it to me once, but I'm not sure anymore.) The protocol is based on tissue digestion with pronase and collagenase in phosphate buffer and extraction in ethanolic ammonia. I can send you a pdf separately.
Finally, I have a jar of barbital and would love to send you some, but let's not get ourselves in trouble!
Cheers and good luck,
Peter
Article Effects of persistent organic pollutants on the thyroid func...
Thanks very much for your answers! This is very helpful information.
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