Lipid and protein analysis using the Direct Detect spectrometer
Analysis of protein and lipid content from a single sample measurement using the Direct Detect spectrometer. Because the sample composition was unknown, analysis of the phospholipids was performed using the “Relative Absorbance” mode. (click to enlarge)

Simplified analysis of lipid or detergent content in biological samples using the IR-based Direct Detect Spectrometer

Lipids account for the major (~50%) compositional and structural element of biological membranes, and are present in many cell-derived biological samples, lysates and/or protein preparations. The Direct Detect® spectrometer enables simultaneous protein quantitation and lipid analysis, in the same sample. This method can be used to monitor the efficiency of detergent removal during preparation of samples for downstream analysis and for quantitation of known lipid(s) in cases where a viable standard curve has been determined.

Traditional techniques for lipid characterization: disadvantages

Analytical characterization of lipids typically requires that samples undergo laborious, multistep preparative processes prior to analysis. Lipids can be isolated by liquid–liquid extraction and separated into classes, often derivatized, and then analyzed. Conventional methods of analysis include measuring iodine value, elemental phosphorus, acid value (saponification equivalent), peroxide value and radiochemical techniques. More recently, classical methods have been replaced by thin-layer chromatography (TLC), gas chromatography (GC), and high performance liquid chromatography (HPLC) as well as mass spectrometry (MS).

Benefits of using Direct Detect Spectrometer for lipid or detergent characterization

The Direct Detect spectrometer uses the C-H symmetric stretching vibrational population between 2870 and 2840 cm-1 to determine lipid or detergent content in a single step, with minimal sample preparation required. The Direct Detect assay-free sample card enables analysis of aqueous-based biological samples, which are normally not compatible with infrared spectroscopy, due to their high water content. These assay-free cards are also compatible with many organic solvents. Given that each lipid possesses an IR signature uniquely defined by its chemical composition and structure, IR spectroscopy further offers a means of qualitative lipid discrimination.
Monitor lipid profile with the Direct Detect Spectrometer
Lipid content as determined using the Direct Detect spectrometer in the “Relative Absorbance” mode in 3 different fractions of breast cancer tissue lysates prepared with CytoBuster™ Protein Extraction Reagent or RIPA buffer. (click to enlarge)

Monitoring lipid profile during lysate preparation

Direct Detect spectrometer has enabled rapid analysis of total protein with simultaneous monitoring of lipid content, offering a means for in-line process optimization for maximal yield and/or purity, and thereby simplifying and improving downstream analysis.