Protein Extraction

Extraction, quantification and analysis of native proteins are fundamental steps in proteomics research. However, protein extraction from in vitro and in vivo systems can pose a serious challenge due to protein heterogeneity and the resistance of the cytoplasmic and nuclear membrane to rupture. Fermentas now offers two new products for efficient protein extraction from cultured cells or tissues: ProteoJET™ Mammalian Cell Lysis Reagent for one-step total protein extraction and ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit for simultaneous isolation of nuclear and cytoplasmic protein fractions from the same sample. Both kits use a mild detergent-based lysis step that permits extraction of native proteins suitable for common proteomics and enzymatic activity assays including immunoprecipitation, affinity purification, 1D and 2D SDS-PAGE, Western blotting and electrophoretic mobility shift assays (EMSA). Proteins extracted with these kits are also compatible with Bradford and other quantification assays.

Protein Quantification

The Bradford assay is one of the most convenient and popular protein quantification procedures as it allows fast, sensitive and accurate results. Fermentas offers a ready-to-use Bradford Reagent. Protein concentration is determined by comparing the spectrophotometric absorption value of the sample to the standard curve values obtained with protein solutions of a known concentration. The accuracy of protein quantification depends on the purity of the proteins used to create the standard curve. Fermentas offers a choice of two new protein standard solution sets: Bovine Serum Albumin Standard Set and Bovine Gamma Globulin Standard Set . Both are composed of highly purified proteins at seven ready-to-use concentrations, eliminating laborious dilution steps. In addition to these products, our bovine serum albumin (BSA)-based Protein Standard Solution is a universal standard for colorimetric determination of protein concentrations by various methods including Lowry and Bradford assays.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE is the most widely used method for characterization of proteins and evaluation of their molecular weights and purity. Nearly all proteins are soluble in the presence of the anionic detergent sodium dodecyl sulfate (SDS). SDS binds to proteins with high affinity which confers a negative charge to the polypeptides and relaxes higher order protein structures. Complete disruption of tertiary and quaternary protein structures is essential to achieve protein separation according to molecular weight. This is accomplished by reduction of the inter- and intra-molecular disulfide bonds of polypeptides by the addition of agents such as dithiothreitol (DTT) or 2-mercaptoethanol and subsequent heating. Our new DualColor™ Protein Loading Buffer Pack and classical Protein Loading Buffer Pack contain all the necessary components for the preparation of protein samples for SDS-PAGE analysis. The DualColor™ Protein Loading Buffer Pack has two tracking dyes: blue and pink. The pink dye assists with monitoring protein migration during Western Blot transfers as it remains visible on the membrane following transfer from the gel.
SDS-PAGE normally is performed in Tris-glycine-SDS electrophoresis buffer. However, for separation of small proteins and peptides, Tris-tricine-SDS electrophoresis buffer is recommended.

Native Protein Electrophoresis

Non-denaturing electrophoresis is used to analyze proteins while preserving native structure and conformation. Under native conditions, the electrophoretic separation of proteins depends on their size, shape and charge. Native protein electrophoresis is often carried out in Tris-glycine electrophoresis buffer. Protein samples used for native electrophoresis should be devoid of strong denaturants such as SDS.

Estimation of Protein Molecular Weight: Protein Ladders and Marker

Fermentas offers both Prestained Protein Ladders and Markers and Unstained Protein Ladders and Marker to assist in the determination of protein molecular weights. We recommend using unstained protein standards for precise determination of molecular weights in any buffer system. Prestained standards are recommended for monitoring the progress of the electrophoresis run and the efficiency of protein transfer to membranes during Western blot procedures. The quality of Fermentas unstained protein MW standards is confirmed by analysis on an Agilent 2100 bioanalyzer (see Fig. 1).

Variation in Protein Mobility

The apparent molecular weights of our prestained protein standards are calibrated in classical Tris-glycine-SDS Laemmli system. However, the prestained protein may have different mobility in other electrophoresis buffer and gel systems. Information on the migration pattern of a particular prestained protein standard on various gel types is provided below:

Spectra™ Multicolor Broad Range Protein Ladder (in pdf, 213 KB)

PageRuler™ Plus Prestained Protein Ladder (in pdf, 193 KB)

PageRuler™ Prestained Protein Ladder (in pdf, 213 KB)

Modifications to natural proteins such as phosphorylation and glycosylation may alter protein mobility. The molecular weights of modified proteins do not always correspond to those of unmodified standard proteins of the same size.

Protein Visualization

Proteins can be detected either within the polyacrylamide gel matrix or after their transfer to a polymeric membrane. Fermentas PageBlue™ Protein Staining Solution is based on the Coomassie Brilliant Blue G-250 dye and is recommended for fast, convenient gel and PVDF membrane staining. It detects as little as 5 ng of protein, and does not require a destaining step. For higher sensitivity staining and detection down to 0.05 ng of protein per band, Fermentas offers the new PageSilver™ Silver Staining Kit.

Figure 1. Analysis of PageRuler™ Unstained Protein Ladder:
A – on the Agilent 2100 bioanalyzer and Protein 200 Plus LabChip® Kit
B – on a 8-16% Tris-glycine SDS-PAGE