I am working with microbial protein samples extracted from mouse gut microbiome samples. These mice were fed 13C algal protein (Sigma 642878) which is pigmented so the samples contain pigment, which has been interfering with the assays I use to quantify my samples (Pierce BCA assay for proteins and pierce colorimetric assay for peptides). I am trying to find a quantification assay that won't be confounded by the pigment-- so it probably shouldn't use color or light absorbance to quantify the concentration. Does anyone have any recommendations, or know if such an assay even exists?
Thank you in advance if anyone has any tips!
Quantifying protein or peptide samples that contain pigments can be challenging due to interference from the pigments in standard assays. Here are some approaches based on the literature:
1. Mass Spectrometry (MS): Liquid chromatography-mass spectrometry (LC-MS) is a powerful method for quantifying peptides in complex samples, including those with pigments. It allows for the separation of peptides from interfering substances and provides accurate quantification without the need for extensive sample purification.
2. Detergent-Based Solubilization: Using detergents like SDS can help isolate proteins from pigments. For example, SDS-PAGE combined with spectrophotometric analysis has been used to separate and quantify proteins in pigment-containing samples.
3. Spectrophotometric Adjustments: If using colorimetric assays like the Bradford assay, pigments may interfere with absorbance readings. In such cases, alternative wavelengths or fluorescence-based assays can be used to minimize interference.
4. Protein Fractionation: Techniques like sucrose density gradient centrifugation can separate proteins from pigments, enabling more accurate quantification.
These methods can be tailored depending on the specific characteristics of your sample and the pigments involved.
Hello if the pigmentation has infulance on protein quntifiation the spectomerty in this case isuseless, beter to use chromatorgaphy technique like HPLC for quantifiaction of protein content afeter separation.
Hi Sadie, pigments will confound colorimetric/UV assays. You’ll get the most reliable results by:
- Removing the pigment before quant;
- Switching to fluorescence- or chemistry-based methods that don’t read in the pigment’s absorbance range;
- Doing absolute quant (amino-acid analysis or LC–MS with standards)
You could include controls:
- Pigment-only blank
- Standard addition
- Dilution linearity
- SDS-PAGE against a protein standard ladder
Hope this is helpful!
The answer to this question is provided by MedChemExpress Technical Support.
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