Proteins and Peptides
The assay is fairly tolerant of salts and buffers in the millimolar range. Samples that are submitted in very dilute amounts might need to be concentrated which will also concentrate those buffers until they possibly begin to interfere with the derivatization chemistry which tags the amino acids with a chromophore immediately before injection. For this reason, we prefer that samples be submitted in the 0.5 to 2 mg/ml range. Only a few things are problematic in the assay. They are:
- Non-volatile amines (TRIS is not acceptable in sample matrices)
- Glycerol (in any concentration)
- Large amounts of Lipids
- reducing sugars and sucrose interfere if the sample needs hydrolysis
If possible, water is the best matrix for sample submission. The sample can also be sent dried down if it is known that it does not have solubility or sticking problems.
For the quantitation, three replicates of 5-10 ugms each (15-30 ugms total) is the best scenario. And if the sample is in a concentration of approximately 0.5-2 ugm/ul this allows accurate pipetting while not adding a lot of buffer to the sample. We know that it is not always possible to submit this much sample and good results can be obtained with less. Below 5 ugms of sample, call first so that a plan can be made to assure useful data. With our flourometric detector, we can see picomoles of amino acids. However, background contamination becomes more and more problematic the lower the amount and makes interpretation of the data difficult.
Alternatively, we can analyze amino acid composition from proteins that are electroblotted onto a hydrophobic support membrane.. Tryptophan and Cysteine cannot be quantitated from PVDF. If these amino acid quantities are required, the sample should be submitted in a liquid form. Samples (> 5µgm per lane) that are prepared by electrophoretic procedures (SDS-PAGE, non-denaturing PAGE, 2-D PAGE) should be blotted to polyvinylidene fluoride (PVDF). Nitrocellulose and nylon membranes are not compatible with hydrolysis. Electroblotting must be performed in non-Tris, non-glycine-containing buffers. The electroblotted proteins can be stained with Coomassie Brilliant Blue R-250 or Ponceau S. After destaining, the sample can be excised from the membrane using a razor blade and washed extensively by vortexing with HPLC grade water in a clean Eppendorf-style test tube. As PVDF membranes contain some number of contaminants themselves it is required that the client supply a blank piece of stained/destained PVDF membrane to act as a subtractive background control. Usually, several lanes of the target sample are needed for successful amino acid analysis. We estimate that ~50% of the sample loaded on the gel will finally be blotted to the PVDF. If you need blotting or staining protocols, recipes or advice you may press HERE or call us at the PCL.
Feed and Solid Samples
Many different types of solid samples can be acid hydrolyzed directly in the liquid HCl. Samples should be ground sufficiently finely to assure a homogeneous sampling. About 100 mgs is a good sample size for this assay however, the assay can be scaled down to ~5 mg samples. Tryptophan and cysteine are not determined in this liquid hydrolysis assay.
Free Amino Acids
Free amino acids can be quantitated. The matrix in which they are submitted should be free of large amounts of proteins, lipids and carbohydrates. The assay works well with serum which has been spun through a molecular weight 5000-6000 cutoff filter. Some free amino acids that are routinely assayed are glutamine, asparagine, citrulline, B-alanine, taurine, tryptophan and ornithine.
We are willing to try to develop assays for unusual amino acids and have some standard protocols on hand. Please drop in or call us and discuss your particular needs.