AllPrep Bacterial DNA/RNA/Protein Kit

AllPrep Bacterial DNA/RNA/Protein Kit
Isolate total nucleic acids and cellular proteins from bacterial cultures.
Size/Component Catalog #
50 preps 47054
Product Description

Details

  • 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.

Specs

Specs

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

SDS/Protocol
Data

Data

 

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)
  Translation regulator activity (GO:0045182) 12 
  Electron carrier activity (GO:0009055)
  Protein binding transcription factor activity (GO:0000988)
  Enzyme regulator activity (GO:0030234)
  Catalytic activity (GO:0003824) 350 
  Receptor activity (GO:0004872)
  Nucleic acid binding transcription factor activity (GO:0001071) 24 
  Antioxidant activity (GO:0016209)
  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