Robust extraction workflow solutions for cells and tissues
Adaptive Focused Acoustics®(AFA) is an advanced computer-controlled technology unique in its ability to control the amount of energy delivered to samples, such as cells. By controlling the dosage of acoustic energy delivered, it is possible to gently disrupt the cell membranes of mammalian cells (e.g., GPCR assays), or abruptly disrupt the cell walls of bacteria (e.g., total protein extraction). Cell lysis differs greatly depending on the species the cells are derived from (e.g., plant cells are significantly harder to lyse than mammalian cells). For hard and soft tissue samples, the cryoPREP® can be coupled with AFA to improve the recovery of targeted biomolecules, such as nucleic acids, proteins, and small molecules.
Diverse Downstream Applications
A. Direct cell lysis
B. Dry tissue pulverization extraction workflow using cryoPREP and AFA
Buffer is a Critical Component
Buffer choice is a critical component for the sample preparation of primary cells and tissues. Traditionally, a buffer is selected based primarily on its lysis properties and secondarily on its biomolecule stabilization properties. Unfortunately, many protocols require harsh chemical and/or extreme physical conditions to achieve lysis, which adversely affects the quality and recovery of target biomolecules. An ideal system would initially stabilize the class of biomolecule to be extracted without a harsh and/or extreme condition. For example, a system with a rapid heat transfer from the sample to a temperature where hydrolytic enzymes are effectively inactivated, both recovery and quality of biomolecules (especially labile biomolecules such as mRNA and phosphorylated proteins) is improved. With the flash sample freezing in the thin-walled tissueTUBE followed by dry pulverization, the surface area of a sample can be dramatically increased. For example, with a 2 mm cube of tissue if converted to a 2 µm cube via the cryoPREP, the surface area increases one million fold. This increased surface area reduces the distance a stabilizing extraction buffer need to diffuse. As the Covaris lysis process is independent of buffer choice, the most appropriate buffer for the downstream analytical method is selected. The direct cell lysis and tissue processing workflows enabled by Covaris uniquely provide scientists with the flexibility to select buffers that satisfy the requirements for traditional and custom-based assays
Cell Lysis powered by AFA
The controlled mechanical energy provided by AFA improves the efficiency of cellular lysis independent of buffer constituents. Therefore, AFA provides the flexibility to homogenize cells and efficiently extract proteins, RNA, DNA, Metabolites, or other targets in buffers optimized for downstream analytical methods.
While some denaturing is tolerable, I have disulfide bonds in my protein that I do not want to disrupt. Will truXTRAC Protein Extraction Buffer DF leave disulfide bonds intact?
Protein buffer DF does have 8 M urea. This buffer will break the disulfide bond to a certain extent. However, you can always evaluate it with buffers such as Super B, which will help in maintaining the three-dimensional structures.
I am interested in extracting intact proteins for downstream functional assays. Functional assays involve a co-factor stain that can be used to check for protein presence by SDS-PAGE and if protein is strep tagged, it can be detected by anti-strep antibody on western blot. Will truXTRAC Protein Extraction Buffer SuperB affect this functional assay?
truXTRAC Protein Extraction Buffer SuperB has non -detergent sulfobetaine(NDSB) which helps in renaturing of the protein. If you are interested in functional assay, please consider using buffer N.,
What non-ionic detergents work well with AFA?
Does Covaris provide an alternative for detergent containing buffer for LC/MS analysis?
The Covaris Protein Extraction buffer DF is a detergent-free buffer that efficiently extracts proteins and compatible with LC/MS analysis.
What is the best buffer to increase total protein yields?
Protein Extraction buffer TP extracts more total protein at lower detergent concentrations in conjunction with AFA. The buffer contains urea and thiourea, and to zwitterionic detergents.
Describe the most suitable buffer for recovering proteins in native conformation?
Covaris Native Protein Extraction Buffer N is a physiological buffer optimized for extracting proteins in their native conformation and is directly compatible with immunoassay, protein assay and affinity enrichment, and any other application requiring proteins in their native conformation.
What is the best buffer for extracting denatured protein?
truXTRAC Protein Extraction Buffer TP or Buffer MJ are recommended for the extraction of denatured proteins.
Can I take extracted protein in Buffer TP or Buffer MJ for direct analysis by MS?
If you are doing LC/MS, then you may inject the sample directly. If you wish to perform reduction/alkylation followed by tryptic digestion, then you will want to dilute the protein to decrease the salt concentration.
Do native protein extraction buffers have any percent of organic compounds?
Both protein extraction buffer N and super B are hydrophilic in nature and do not contain any organic compounds.
10. What is the advantage of using truXTRAC Protein Extraction Buffer SuperB instead of truXTRAC Protein Extraction Buffer N
Protein extraction buffer Super B increases protein yield and preserves the native conformation and biological activity. The buffer does not form micelles and prevents protein aggregation; however, protein Buffer N does have protein aggregation in low percentage.
Covaris provides tools and technologies to improve pre-analytical sample preparation, enable novel drug formulations, and manage compounds in the drug discovery process.