- Product Description
- Powerful lysis - Optimized bead beating method enables efficient lysis and solubilization of total nucleic acids and cellular proteins from gram-negative and gram-positive bacteria
- Easy and fast protein extraction - Novel spin filter-based method enables sequential DNA, RNA and protein extraction in less than 40 minutes without the need for precipitation
- Accurate results – Nucleic acids can be used in PCR, qPCR, RT-PCR, and next-generation sequencing; protein can be used for mass spectrometry, 1D and 2D gel electrophoresis
The AllPrep Bacterial DNA/RNA/Protein Kit is designed to isolate total nucleic acids and cellular proteins from bacterial cultures in a user-friendly spin column format. An optimized bead beating method enables efficient lysis and solubilization of nucleic acids and proteins from diverse gram-negative and gram-positive species. The use of silica spin columns to achieve reversible, sequential immobilization of DNA, RNA and proteins greatly streamlines the nucleic acid and protein isolation process, enabling direct correlations between genes, their expression, and function. Isolated nucleic acids (gDNA, rRNA, mRNA, small RNAs) are suitable for the most demanding downstream applications including PCR, qPCR, RT-PCR, and next-generation sequencing. Purified proteins are suitable for 1D SDS-PAGE and mass spectroscopy following in-gel trypsin digestion. Some applications, including 2D SDS-PAGE and solution phase proteolytic digestion for mass spectrometry, may require an additional detergent removal step following protein elution.
This novel sequential isolation method begins with mechanical lysis of cultured bacterial cells using a 0.1 mm glass bead tube. Nucleic acids and proteins are completely solubilized during homogenization and mixed with a DNA binding solution. DNA is bound to a spin column and the flow-through containing RNA and proteins is then combined with a solution that binds total RNA on a second spin column. The final flow-through, containing denatured proteins, is combined with another novel buffer to immobilize the proteins onto the third and final silica spin column. Each spin column containing either immobilized nucleic acids or proteins is then washed and the immobilized analyte is eluted.
For isolation of DNA, RNA and Protein from Fungal cultures, try our AllPrep Fungal DNA/RNA/Protein Kit.
Format Silica spin filter* Method Bead Beating Sample Size 1.8 ml of microbial culture
MOBIO recommends using no more than 1x10^9 bacterial cells per sample
Throughput 1-24 samples Storage We recommend storing spin filters at 4°C. All other kit reagents and components can be stored at room temperature (15-30°C). Bead Type 0.1 mm glass Sample Types Processed Cultured gram-negative and gram-positive bacteria Equipment Required Vortex and Vortex Adapter (13000-V1-24) Time 36 minutes Catalog # 47054, 47050
*Patent pending method for protein extraction
Figure 1. Sequential spin filter binding captures and concentrates DNA (1), RNA (2) and proteins (3) in lysate from gram-negative and gram-positive cultures. 1.8 mL of overnight cultures were processed using the AllPrep Bacterial DNA/RNA/Protein Kit. Nucleic acids were eluted in 100 μl of their respective elution reagent. For (3), proteins were eluted in 100 μl of 1% SDS/HEPES elution buffer. 10 μl of each sample was run on a Bio-Rad Any kD™ Mini-PROTEAN TGX Stain-Free™ Gel for 25 minutes at 200V. Gels were stained using SimplyBlue SafeStain. A: E. coli; B: E. faecalis; C: P. aeruginosa; D: S. aureus; E: B. subtilis.
Table 1. High nucleic acid and protein yields (100 µl elution volume) Sample DNA concentration (µg/ml) RNA concentration (µg/ml) Protein concentration (mg/ml) E. coli 103 668 2.36 E. coli 114 669 2.25 E. faecalis 72 264 0.987 E. faecalis 57 234 1.28 P. aeruginosa 130 571 1.38 P. aeruginosa 131 573 1.59 S. aureus 53 263 1.35 S. aureus 61 267 3.34 B. subtilis 55 233 1.53 B. subtilis 66 251 1.54
Figure 2. Successful 2D SDS PAGE analysis of E. coli proteins extracted using the AllPrep Bacterial DNA/RNA/Protein Kit. 1 ml of overnight culture was processed using the AllPrep Bacterial DNA/RNA/Protein Kit. Eluent was run, without modification, on a pH 4-8 IEF gradient and 10% acrylamide slab gel using an SDS-compatible carrier ampholine (CA). Molecular weight standards (220,000, 94,000, 60,000, 43,000, 29,000, and 14,000). 2D analysis was performed by Kendrick Labs, Madison, WI.
Figure 3. Diverse RNA transcript sizes isolated from E. coli using the AllPrep Bacterial DNA/RNA/Protein Kit. 1 mL of overnight culture was processed using the AllPrep Bacterial DNA/RNA/Protein Kit. Total RNA was assayed using the Affymetrix GeneChip E. coli 2.0 Genome Array. Only expression data with a mean fluorescence intensity over 100 (3X background) was considered.
Table 2. Diverse RNA species isolated from E. coli using the AllPrep Bacterial DNA/RNA/Protein Kit.
Molecular Function # of Genes/Function Transporter activity (GO:0005215) 45 Nutrient reservoir activity (GO:0045735) 1 Translation regulator activity (GO:0045182) 12 Electron carrier activity (GO:0009055) 1 Protein binding transcription factor activity (GO:0000988) 1 Enzyme regulator activity (GO:0030234) 3 Catalytic activity (GO:0003824) 350 Receptor activity (GO:0004872) 3 Nucleic acid binding transcription factor activity (GO:0001071) 24 Antioxidant activity (GO:0016209) 5 Structural molecule activity (GO:0005198) 44 Binding (GO:0005488) 119
Figure 4. High quality total RNA from E. coli using the AllPrep Bacterial DNA/RNA/Protein Kit. 1 ml of overnight culture was processed using the AllPrep DNA/RNA/Protein Kit. 2 µL of RNA eluent was loaded into an Agilent TapeStation. RNA Integrity numbers (RIN) were 10 and clearly showed small RNAs present. Average RNA concentration loaded was 345 ng/µL.
Figure 5. Equivalent mass spectrometry (MS) results with the AllPrep Bacterial DNA/RNA/Protein Kit and conventional protein extraction methods. The AllPrep Bacterial DNA/RNA/Protein Kit was compared against a conventional protein extraction method, sodium carbonate, via MS. E. coli cells were resuspended in sodium carbonate and subjected to bead beating. The crude, unpurified lysate was run alongside isolates processed using the AllPrep Bacterial DNA/RNA/Protein Kit. 20 μg of protein isolate was run on a Bio-Rad Any kD™ Mini-PROTEAN TGX Stain-Free™ Gel for 25 minutes at 200V. The gel was stained using SimplyBlue SafeStain and the section contained within the indicated square was excised and analyzed by MS (A). Identified proteins with >4 peptide matches were classified using PANTHER version 10. The results indicated comparable protein class isolation between both samples (B). Mass spec analysis by MS Bioworks, LLC.
Table 3. Comparable MS results to traditional methods. AllPrep Bacterial DNA/RNA/Protein Kit Sodium Carbonate Method Total number of proteins identified 384 388 Total number of spectra matching 5585 5433 Total number of unique peptides 4005 3931