• Eliminates artifacts caused by incompatible contaminants –
  removes dyes, reducing agents, detergents, sugars, glycerol, guanidine, urea and ammonium sulfate to provide reproducible results on SDS-PAGE analysis
• Compatible with the BCA Assay  – allows quantification of the processed sample
• Enriches dilute protein solutions – concentrates protein sample by eight-fold in less than 20 minutes for SDS-PAGE analysis
• Fast and easy-to-use for up to 70 µg of protein per sample – uses new spin cup format that allows higher amounts of protein to be processed than with the original procedure

The reformatted Pierce SDS-PAGE Sample Prep Kit improves SDS-PAGE analysis of difficult-to-analyze samples. While numerous compounds are incompatible with typical electrophoretic running buffers and SDS-PAGE separation, the Pierce SDS-PAGE Sample Prep Kit cleans up protein samples in minutes, removing a wide range of interfering chemicals, such as chaotropic agents, detergents, lipids, pH extremes and salts.

For example, mixing protein samples containing 6 M guanidine•HCl with Laemmli buffer results in precipitation. High concentration of detergents often result in distorted bands during SDS-PAGE analysis (Figure 1). Treatment with the Pierce SDS-PAGE Sample Prep Kit can eliminate many interfering agents that are difficult to remove by standard sample-processing methods. Use of this treatment will result in reliable SDS-PAGE analysis.

Figure 1. Eliminate distortion caused by detergents.

Rat C6 cells were lysed and a membrane protein fraction isolated using Mem-PER Eukaryotic Membrane Protein Extraction Reagent (Product # 89826). Membrane and hydrophilic cell fractions were separated by SDS-PAGE using 4-20% gradient gels with or without prior treatment using the PAGE-prep Resin. Western blot analysis was performed using an antibody against cytochrome oxidase subunit 4 (COX 4) and SuperSignal West Femto Chemiluminescent Substrate (Product # 34095). Pierce SDS-PAGE Sample Prep Kit-treated samples exhibit better band straightness and resolution with low molecular weight proteins than samples that were untreated.

Sample concentration is also critical for proper SDS-PAGE analysis. Protein samples that are too dilute to be detected reliably can be rapidly concentrated using the PAGEprep Advance Kit and directly analyzed by SDS-PAGE.

S = Soluble fraction (hydrophilic)
M = Membrane fraction

To use the kit, 2 - 300µl of a sample containing up to 70 µg of protein is combined with 20 µl of PAGE-prep Advance Resin, which is composed of modified diatomaceous earth. Added DMSO enables all proteins to bind to the resin, while unbound contaminating chemicals are washed through using the provided spin cups. Proteins are released with 50 µl of Elution Buffer, which is compatible with the BCA Protein Assay, allowing quantitation of a portion of the processed sample, if desired. The eluted sample can then be combined with the supplied 5X Sample Buffer or alternative sample buffers and analyzed by SDS-PAGE.

The optimized spin cup format and Pierce SDS-PAGE Sample Prep Kit procedure is more efficient and faster than the original PAGEprep Kit’s batch method, reducing sample loss while improving recovery, consistency and capacity. Complete processing of samples can be conducted in less than 20 minutes. Approximately 70-85% of the sample is recovered for most proteins when processing 1-70 µg, as recommended in the procedure.

The Pierce SDS-PAGE Sample Prep Kit prepares the following sample types for SDS-PAGE analysis:

Inclusion bodies solubilized in guanidine•HCl

Samples containing low-pH buffers, thiocyanate or urea

Proteins precipitated in ammonium sulfate

Dilute protein solutions

Figure 2. Consistent protein recovery is achieved using the Pierce SDS-PAGE Sample Prep Kit. Pure proteins (60 µg) of assorted molecular weights: 30K, 44K, 80K, 86K and 12K and bacterial lysate at 27K were processed using this kit. Protein concen-trations were determined with the BCA Protein Assay and reported as percent protein recovered.