- Product Description
- Powerful lysis - Optimized bead beating method enables efficient lysis and solubilization of total proteins from fungi, gram-negative and gram-positive bacteria
- Easy and fast protein extraction - Novel spin filter-based method enables protein extraction in just 22 minutes without the need for precipitation
- Accurate results - Protein can be used for mass spectrometry, 1D and 2D gel electrophoresis and other proteomics applications
The NoviPure Microbial Protein Kit is designed to isolate total cellular proteins from microbial cultures in a quick and user-friendly spin filter format. An optimized bead beating method enables efficient lysis and solubilization of total proteins from a large diversity of microbial species including fungi, gram-negative and gram-positive bacteria. The use of silica spin filters to achieve reversible immobilization of proteins — a patent-pending technical advance in protein extraction — greatly simplifies the isolation process by removing traditional cumbersome and bias-inducing precipitation steps. Isolated proteins are suitable for many downstream applications such as 1D and 2D SDS PAGE as well as mass spectrometry.
This novel protein isolation method begins with mechanical lysis of cultured microbial cells using a mixed glass bead tube. Proteins released during homogenization are denatured and solubilized by a strong chaotropic agent. An optimized two-step binding process is then used to immobilize all solubilized proteins onto a silica spin filter. In the first step, a salt solution is added to the microbial lysate and passed over a silica spin filter to bind most proteins. In the second step, the flow through is adjusted with a second salt buffer and passed through the same spin filter. This second salt buffer promotes low molecular weight protein association with the silica spin filter. Immobilized proteins are then washed and finally eluted.
Format Silica spin filter* Method Bead Beating Sample Size 1.8 ml of microbial culture
MOBIO recommends using no more than 1x10^8 fungal cells or 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, 0.5 mm glass mix Sample Types Processed Cultured fungi, gram-negative and gram-positive bacteria Equipment Required Vortex and Vortex Adapter (13000-V1-24) Time 22 minutes** Catalog # 47044, 47040
**Not including optional DNase/RNase treatment
Figure 1. Spin filter binding captures and concentrates proteins in lysate from fungal, gram-negative and gram-positive cultures. 1 ml of overnight culture was processed using the NoviPure Microbial Protein Kit. Two samples from each culture 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. Crude, unpurified lysate from each culture was used as a control to demonstrate that the NoviPure spin filter captures and concentrates the majority of proteins present in the microbial lysate from fungal (A), gram-negative (B) and gram-positive (C) species. PS: Protein standard; C: Crude, unpurified lysate for each culture. Table 1. High protein yields are achieved with the NoviPure Microbial Protein Kit. Sample Protein Yield (mg/ml) E. coli 1 9.38 2 8.65 3 8.45 Average 8.83 C. albicans 1 4.42 2 4.81 3 4.27 Average 4.50
Figure 2. Successful 2D SDS PAGE analysis of E. coli proteins extracted using the NoviPure Microbial Protein Kit. 1 ml of overnight culture was processed using the NoviPure Microbial Protein Kit as described in Figure 1. 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. Equivalent mass spectrometry (MS) results with the NoviPure Microbial Protein Kit and conventional protein extraction methods. The NoviPure Microbial 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 NoviPure Microbial 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). The results indicated comparable protein family isolation between both samples (B).
Table 2. Number of proteins identified, matching spectra and unique peptides is comparable for the two methods.
NoviPure Microbial Protein Kit Sodium Carbonate Method Total number of proteins identified 417 384 Total Number of spectra matching 5527 5585 Total number of unique peptides 4041 4005