E.g. amino acid evaluation [38], size exclusion chromatography or mass spectrometry
E.g. amino acid evaluation [38], size exclusion chromatography or mass spectrometry are usually linked to a high instrumental expense and work in preparatory perform [14]. The higher protein specificity with the latter procedures is advantageous for target protein quantification. Having said that, the identical, high sensitivity towards diverse types of proteins and also the related will need to work with acceptable requirements are significant drawbacks within the context of total protein quantification. In case of techniques that happen to be much less distinct, e.g. UV/ Vis-based platform Cadherin-3, Human (630a.a, HEK293, His) SoloVPE [30] instrumental advances have allowed for a rise in sensitivity and lower of your sample volume. Nevertheless, protein Tryptophan Hydroxylase 1/TPH-1, Human (His) quantification through UV/VIS absorption is normally hindered by sample matrices containing unsaturated fatty acids [47]. In colorimetric assays, lab-on-a-chip systems [2, 14, 35] have led to substantial progress when it comes to sensitivity and reproducibility. These methods combine a chromatographic separation phase for the colorimetric detection step, leading to great resolution and sensitive quantification. On the other hand, the chromatographic separation step has to be especially adapted towards the sample matrix. In case of bioprocess samples this sample matrix might be subjected to dynamical changes more than course of action time. This needs case-by-case adaptations of the chromatographic separation step and tends to make total protein quantification by way of such systems tedious. On top of that, owing to the need for advanced microfluidics in the chip technologies, these assays are linked to substantial investments and larger consumable costs as compared to conventional colorimetric assays. Wet-chemical assays are additional price efficient and, even though involving quite a few handling and preparation actions, typically enable for high-throughput analysis. The underlying principle of a extra or less uniform protein staining, based merely on amino acid residues, is an advantage within the context of total protein quantification. In mixture with their simplicity the latter qualities would be the cause for the wide usage of those wet-chemical assays for totalprotein quantification [6, 13, 29, 32, 47]. Bradford, Lowry and the Bicinchoninic acid (BCA) assay are the most commonly made use of colorimetric assays. Specifically in microbial bioprocesses the composition with the supernatant sample normally becomes increasingly complex all through the fermentation time course, mostly as a result of a gradual accumulation of sugars, phospholipids, DNA and salts. Considerable investigation effort has been devoted to the direct comparison from the out there colorimetric assays, leading to some common suggestions regarding assay usage [11, 22, 29, 37]. The Bradford or Coomassie Blue assay is based on a residue-specific stain, very first described by Bradford [4]. Via hydrophobic interactions, Coomassie Brilliant Blue G-250 [11] binds to arginine, histidine, phenylalanine, tryptophan and tyrosine residues [8] at acidic pH. Disadvantages of this assay contain sensitivity to different reagent formulations [33] too because the high sensitivity to varying amino acid composition [8]. This sensitivity for the amino acid distribution renders the method less applicable for the generic quantification in the total protein content in biotechnology. The Lowry assay is based on a two-step chemical reaction: first, a reduction of cupric ions to cuprous ions below alkaline circumstances, and second, a reduction of protein residues [24]. This reduction is followed by a reaction using the Foli.
