Plant Protein Extraction Kits

• Tested and validated on most commonly studied plant species — Arabidopsis, maize, soybean, cotton, tobacco, pine needle and more
• Tested and validated on tissues – leaf, flower, root and seedlings
• Extracts with high specificity for protein fractionation
• Provides highly reproducible results for data comparison
• Two-dimensional electrophoresis compatible
• Performs over a wide sample prep dynamic range

Tested on Multiple Species and Tissues

Figure 1  Total proteins were extracted from multiple plant species and tissues using Plant Total Protein Extraction Kit, then separated on 10–20% Tris Glycine SDS-PAGE gels. Detection of proteins was accomplished by staining the gel with ProteoPrep silver staining kit. Lanes 1–7 are leaf tissue, Lanes 8 & 9 are flower tissue, Lane 10 is seedling tissue, Lane 11 is stem tissue and Lanes 12–14 are root tissue. Lanes and Species are indicated below: 1) Arabidopsis 2) Tomato 3) Tobacco 4) Soybean 5) Maize 6) Cotton 7) Pine Needle 8) Arabidopsis 9) Tobacco 10) Arabidopsis Seedling 11) Soybean 12) Maize 13) Soybean 14) Tobacco 15) Markers

Plant Hydrophilic Protein Extraction Kit and Plant Hydrophobic Protein Extraction Kit Provide Specific Protein Fractionation

Figure 2  To show specificity of protein fractionation, extracts from Arabidopsis thaliana leaf rosette tissue were analyzed by Western blot SDS-PAGE immuno transfer analysis. Sequential protein extracts of leaf rosettes employing Plant Hydrophilic Protein Extraction Kit and Plant Hydrophobic Protein Extraction Kit were separated on 4–12% Tris-Glycine SDS-PAGE gels. Proteins were transferred to Immobilon-P membranes. Membranes were blocked with 3% Bovine serum albumin in Tris buffered saline containing 0.05% Tween-20, pH 8.0. Transferred protein were immunoreacted with Arabidopsis specific primary antibody markers and secondary peroxidase antibody conjugates. Detection of secondary conjugates was accomplished using peroxidase colorimetric substrate 3,3',5,5'-Tetramethylbenzidine (TMB). Gel A. Hydrophilic soluble Ribulose Bisphosphate Carboxylase/ oxygenase large subunit (RbcL) is detected in the fraction from the Plant Hydrophilic Protein Extraction Kit (1:5000 Ck anti-Rubisco, 1:16,000 Rb anti-Ck IgY HRP). Gel B. Hydrophobic Chloroplast Thylakoid Membrane Protein, Chlorophyll a/b binding protein (CAB) is detected in the fraction from the Plant Hydrophobic Protein Extraction Kit (1:1000 Rb anti-CAB Chlorophyll a/b binding protein, 1:16,000 anti-Rb (wm) HRP).

Reproducible Results Enable Data Comparison

Figure 3  Protein extracts of young tomato leaf were prepared following fractionation protocols. Bradford protein determinations were made and dilution to 0.03 mg/ml samples were prepared in LDS sample buffer with 25 mM DTT. Extract sample loads of 15 µl (0.45 µg) were separated on NuPAGE 4–12% BisTris 1.0 mm gels. Protein bands were detected using ProteoSilver Plus Silver stain kit. Gel A. Triplicate extracts from the same tissue sample of plant 1. Gel B. Extracts from two separate tissue samples of plant 1. Gel C. Extracts from two separate tissue samples of plants 2 & 3. Predominant bands are present for each extraction.

Two-Dimensional Electrophoresis on Plant Protein Extracts

Figure 4  Hydrophilic protein extract of young Arabidopsis thaliana leaf rosettes was obtained using Plant Hydrophilic Protein Extraction Kit following tissue disruption by grinding in liquid nitrogen. Protein sample was reduced with tributylphosphine and alkylated with iodoacetamide. Sample (25 µg) was loaded on a 7 cm IPG strip, pH 4–7 and allowed to rehydrate overnight. Proteins were separated in the first dimension using a step voltage gradient totaling 2300 Volt hours maintaining less then 50 µA, 0.1 Watt per strip. Separation in the second dimension were by 4–20% Tris Glycine Zoom SDS-PAGE gel at 40 mA, 15 Watts, 190 volts. Detection of protein spots was accomplished by staining the gel with SYPRO Ruby protein gel stain and the gel was imaged with a BioRad Fluoro-S MultiImager.

Excellent Performance over a Wide Range of Sample Load

Figure 5  Total proteins were extracted from young tomato leaf tissue. Tissue samples from 270, 140, and 10 mg were extracted and separated on 4–20% Tris Glycine SDS-PAGE gel. Detection of proteins accomplished by staining gels with ProteoPrep silver staining kit. Gel was imaged on a BioRad Fluro-S MultiImager. Densitometry was performed to compare extraction efficiencies of various tissue amounts. Volumetric lane analysis plot demonstrates equivalance in extraction efficiency throughout the sample preparation dynamic range.