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PCT Dependent Detergent-Free Extraction of Proteins from Lipid-Rich Samples

This kit is intended for the extraction of total protein from mammalian adipose tissues and other high lipid content samples.

For Research Use Only. Not for use in diagnostic procedures.

Barocycler, PULSE Tubes and ProteoSolve are trademarks of Pressure BioSciences, Inc.

Kit Contents ProteoSolveLRS PULSE Tubes™ FT-500 (1.4 mL) Sample Collection Tubes (2.0 mL)

12 each 24 each

Reagent A (Extraction reagent)

20 mL

Reagent B (Partitioning reagent)

20 mL

Reagent C (Precipitation Reagent)

60 mL

Storage o

All kit components should be stored at room temperature (15-30 C) in well-ventilated area. Note:

Extraction reagent possesses strong chaotropic properties and promotes protein denaturation. It is expected that the activity of proteolytic enzymes will be abolished or significantly reduced in samples stored in solution in reagent A (whether or not Reagent B has been used during extraction.)

Note:

Protein pellets may be considered stable if lyophilized or stored frozen. It is suggested that protease inhibitor cocktail (not supplied with the kit) be added to the reconstitution media to prevent possible protein degradation during handling and extended storage of samples following the extraction.

Safety Information Standard laboratory safety practices should be employed when working with chemicals in order to minimize potential exposure to hazardous materials. Reagent A contains hexafluoroisopropanol, which can cause serious eye injuries on contact, and is a strong skin and respiratory irritant. This product may also contain a very low level of hexafluoroacetone, which presents a potential reproductive hazard. Care should be taken to avoid contact by wearing appropriate gloves, safety glasses and other protective equipment as needed. Handle Reagent A in the chemical exhaust hood. For more information, please consult the appropriate Material Safety Data Sheet (MSDS).

Product Use Limitations ProteoSolveLRS is intended for Research Purposes Only. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.

Product Warranty and Satisfaction Guarantee PBI guarantees the performance of all products as described in our product literature. If ProteoSolveLRS does not meet your expectations, please contact our Technical Service

Department. PBI reserves the right to change, alter, or modify any product to enhance its performance and design.

Technical Assistance At PBI we pride ourselves on the quality and availability of our technical support. Our Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample preparation, proteomics, molecular biology and the use of PBI products. If you have any questions or experience any difficulties regarding ProteoSolveLRS or other PBI products or services, please do not hesitate to contact a Technical Service Representative at: [email protected] or by calling Technical Support at 508-580-1818 between 9:00 am and 5:00 pm Eastern Standard Time.

Introduction The ProteoSolveLRS kit is designed for detergent-free extraction of proteins from tissues rich in lipids, such as adipose tissue and brain tissue. The protein extracts from this kit are directly compatible with HPLC separation and LC-MS/MS analysis. Following the removal of excess solvent by evaporation under vacuum or protein precipitation and reconstitution in a suitable aqueous reagent, these extracts are also compatible with SDS-PAGE and 2D electrophoresis applications.

Principle and Procedure Tissue samples are disrupted using Pressure Cycling Technology in Reagent A, which contains an optimized concentration of the amphiphilic organic solvent, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). This reagent is miscible with lipids and aqueous solutions, acting as a volatile solvent with detergent-like properties to keep both hydrophobic and hydrophilic sample constituents in solution, once they are extracted from cells and tissues. The Barocycler™ instrument subjects samples to alternating cycles of hydrostatic pressure, causing destabilization of the cytoskeleton and lipid bilayer, cell disruption and dissolution of the cellular components. Fresh or frozen tissue samples are loaded through the ram side of the PULSE Tube (Figure 1A). The ram is then inserted as far as it can go into the tube until it comes in contact with the tissue sample and presses the sample against or through the lysis disk (Figure 1B). The opposite end of the PULSE Tube comprises the fluid chamber into which up to 1.1 mL of Reagent A depending on the total sample volume and 200 µl of Reagent B (Figure 1C)(total volume of sample plus reagents should not exceed 1.4mL). Volume of reagent B may have to be reduced when tissue samples larger than 100 mg are used. If subsequent analysis of the sample’s lipid composition is required then omit reagent B and substitute the equivalent volume of reagent A. Liquid samples as well as suspensions of small particles or emulsions can be loaded through the cap side together with the extraction reagents AFTER the ram is inserted. After addition of Reagents A and B, the threaded cap is tightly closed using the PULSE Tube tool provided with your Barocycler™ instrument. Prior to placing the PULSE Tube into the Barocycler vigorously mix using a vortex mixer for 10-20 seconds. The PULSE Tube(s) are then inserted into the instrument pressure chamber such that the threaded cap of each tube is facing up. For most samples, processing is performed using the following conditions: 35,000 psi for 20s; ambient pressure for 20s, 20 cycles per run. (Please refer to the Barocycler Instrument manual for more information). Following pressure cycling, remove the PULSE Tubes from the instrument and vigorously mix using a vortex mixer for 10-20 seconds again. Open the threaded cap and

transfer all of the liquid extract out of the PULSE Tubes into the corresponding Sample Collection tubes supplied with the kit. In order to assure complete retrieval of the liquid extract, slowly and carefully push the ram up as far as it can go by pressing the tubes against the PULSE Tube tool supplied with the instrument (Figure 1). If residual tissue pieces are noticeable on or under the lysis disk, a second round of extraction can be performed by addition of fresh Reagents A and B. Transfer liquid extracts into the 2 mL sample collection tubes. Vortex the sample collection tubes again for 20 seconds and centrifuge at 8,000×g for 15 minutes. During the centrifugation extracts will separate into two liquid phases. Warning! Do not exceed centrifugal force over 8,000×g to avoid possible tube damage and sample loss. Note:

For larger (over 200 mg of tissue) samples of extremely lipid-rich tissue (e.g. adipose tissue) Reagent B may be replaced by the equivalent amount of Reagent A to maintain optimal sample/reagent ratio.

The bottom liquid phase of each sample, which contains proteins, DNA, RNA and other hydrophilic compounds should be transferred into a clean sample collection container by aspiration with a suitable fine pipette tip. Subsequent analysis of this fraction may be conducted by one of several methods, as described below. The remaining upper, hydrophobic phase can be collected for the analysis of lipid constituents of the sample or discarded, if such analysis is not intended. It has been shown that the top fraction typically contains little or no extractable protein.

HPLC and LC-MS Compatibility At this stage the extract is ready for fractionation by means of high performance liquid chromatography (HPLC) using Strong Cation Exchange (SCX) or Hydrophilic interaction (HILIC) stationary phase covalently bound to the chromatography support. Please ensure that the stationary phase of the HPLC column you select is compatible with injections of high concentrations of fluorinated alcohols such as 1,1,1,3,3,3-Hexafluoroisopropanol or trifluoroethanol. Chromatographic separation on either SCX or HILIC stationary phase employing a wide variety of traditional elution methods should provide sufficient separation between any residual lipid components (which have a very low retention on column in most protocols) and proteins. The latter can be fractionated by their charge (SCX mode) or hydrophilicity (HILIC) mode into a number of fractions, suitable for subsequent analysis by conventional proteomic methods, including, but not limited to variations of the following: a. enzymatic digestion followed by capillary RP-HPLC and electrospray ionization LC-MS/MS b. solvent removal by evaporation followed by SDS-PAGE electrophoresis/MALDITOF MS c. solvent removal by evaporation followed by 2D electrophoresis/MALDI-TOF MS For general guidelines on solvent removal and reconstitution please refer to the following section of this manual.

Solvent Removal by Evaporation If removal of reagent A is required for further sample processing, the solvent can be effectively evaporated by using a centrifugal vacuum evaporator (e.g. SpeedVac or equivalent), equipped with strong vacuum pump and a suitable refrigerated solvent trap. Please ensure that all the vacuum connections are secure and no latex or butadiene rubber vacuum hoses are used.

Note:

It is advisable to consult with the vendor of your evaporator equipment to confirm its compatibility with fluorinated alcohols such as HFIP or trifluoroethanol.

Note:

Tygon-branded PVC or Teflon vacuum tubing is strongly recommended if evaporation of reagent A is intended to become routine in your laboratory.

The vacuum pump outlet must be properly vented into a chemical exhaust hood. The Solvent o Trap must be turned on and chilled to at least -20 C prior to solvent evaporation. Ensure that the bottom fraction has been cleanly separated from the lipid layer, as the presence of lipids will substantially increase solvent evaporation time. If significant lipid contamination is observed, repeat centrifugation and transfer lower phase in to a clean test tube. Although unlikely, if a residual lipid layer forms during evaporation it can be removed with the thin gel loading pipette tip. During solvent evaporation, proteins and other non-volatile compounds will remain on the bottom of the 2 mL sample collection tube, while the extraction solvent (REAGENT A) is evaporated to a residual volume of 5-10 µL. Proteins can then be brought up in a buffer of choice (e.g. SDS-PAGE sample buffer) for subsequent electrophoretic separation. It is imperative to check the evaporation rate periodically to prevent complete drying of the sample, as this may result in pellets that are difficult to dissolve.

Alternative Solvent Removal: Protein Precipitation Reagent A possesses strong chaotropic properties leading to denaturation of proteins in solution. Since denatured proteins typically exhibit altered solubility, rapid dilution of such solution with certain reagents will lead to the precipitation of the proteins. Extracted proteins can be rapidly precipitated out of Reagent A. Reagent C (Precipitation Reagent) is provided in the kit for your convenience if protein precipitation is intended. Generally, a dilution of sample with a 3-fold excess (by volume) of Reagent C followed by the 10 minute incubation on ice and centrifugation for 15 minutes at 8,000 g is required for efficient protein precipitation. This approach may provide considerable time savings. Additionally, precipitation is not adversely effected by residual lipid carryover and does not require a vacuum concentrator. Several points must be taken into consideration when choosing an optimal solvent removal method: 1. Evaporation may lead to less protein loss. 2. For sample analysis by chromatography following protein extraction, evaporation may not need to be as thorough and complete, since excess Reagent A will be removed during the chromatography analysis. 3. Addition of exogenous salt(s) may be utilized to selectively precipitate certain proteins and keep other protein species in solution in an attempt to stepwise fractionate complex protein mixtures. 4. Incomplete removal of the lipid layer will significantly increase evaporation time, but will not interfere with the precipitation. 5. Precipitation may not be efficient at sample concentrations below 150 mg sample per 1ml of reagent A. For such samples evaporation is recommended. Alternatively, additional amount of Reagent B can be used to decrease the amount of Reagent A used for extraction of a small tissue sample. Precipitation is not affected by the presence of Reagent B layer above the extracted sample.

Analysis of Lipid Composition The top liquid phase will contain lipophilic constituents of the sample. If desired, analysis of the lipid composition of the sample can be performed using GC-MS or HPLC. In this case, the addition of Reagent B should be omitted, as it contains aliphatic hydrocarbon components that may interfere with a suitable analytical method. Instead, desired phase separation may be achieved by using a suitable sample size, at least 100 mg per PULSE Tube for most adipose tissue samples and a suitable amount of Reagent A resulting in a total sample + reagent volume of 1.2 – 1.4 mL per PULSE Tube.

1. Add Sample

2. Set the Ram

3. Add Reagents and Cap

Figure 1. PULSE Tube-a Single Use Sample Processing Vial. Tissue samples are loaded into the tube from the ram side. The Ram is inserted and set to the full extent of its travel into the tube until it is pressing the tissue against the lysis disk. Subsequently the tube is positioned vertically with the threaded cap side pointing upward and the required amounts of extraction solvents are introduced into the chamber of the PULSE Tube. The cap is then closed tightly. The sample is ready to be processed in the instrument

Important Notes:

Determining the Amount of Starting Material It is essential to use the correct amount of starting material in order to obtain optimal protein yield. The maximum amount that can be used is determined by:

• •

The type of tissue and its protein content The volume of solvent required for efficient lysis and extraction

When processing samples, the total volume of starting material and extraction solvent must be maintained between 1.2 mL and 1.4 mL per PULSE Tube. Specifications Specification

Value

Maximum loading volume Minimum loading volume Amount of tissue sample Delipidated extract volume* † Final sample volume Possible downstream Applications

1.4 mL 1.2 mL 20-250 mg 0.5-1.2 mL 10-15 µL

HPLC (SCX or HILIC), isoelectric focusing, SDS-PAGE, Western Blotting

* Varies depending on the sample lipid content Vacuum sample concentrators vary in their performance. A high-efficiency vacuum pump is recommended for rapid solvent evaporation †

Note:

If the maximum capacity for starting material is exceeded, protein yields may be reduced due to inefficient or incomplete lysis.

For further guidelines on using the PCT SPS, refer to the PCT SPS Handbook or Pressure BioSciences Website (http://www.pressurebiosciences.com).

Protocol:

Extraction of Proteins from Mouse Adipose Tissue Using Pressure Cycling Technology (PCT) and the ProteoSolveLRS Kit.

Procedure 1. Determine the amount of tissue to be used. Do not exceed 250mg of sample tissue per PULSE Tube. 2. Place tissue in ram side of PULSE Tube and set the ram. Whole tissue pieces should be loaded into the ram end of each PULSE Tube. Set the ram using the PULSE Tube tool. Add REAGENT A to the fluid retention chamber (sample chamber in Figure 1). The assembled PULSE Tube is then vortexed for 10-20 seconds prior to being loaded into the Barocycler. Program the maximum pressure, number of cycles, time intervals at ambient and high pressure and run the sample(s).

Under certain circumstances, grease from an oily sample (such as adipose tissue), can lubricate the space between the PULSE Tube wall and the ram O-rings. When this occurs, the ram can be pushed out of the PULSE Tube during a depressurization cycle, resulting in sample leakage out of the PULSE Tube. The PULSE Tube Clip is designed to prevent ram extrusion by limiting the movement of the ram. To use the PULSE Tube Clip, slip the hook end beneath the ram and adjust the L-curve to fit between the grooves of the PULSE Tube cap. Place the PULSE Tube with re-usable Lawrence clip into the Barocycler and proceed with PCT.

Note:

If the tissue and buffer have been already added to the PULSE Tube and then frozen for storage, allow the contents to thaw in the PULSE Tube at room temperature (15-30°C).

3. Place samples into Barocycler and initiate pressure cycling. While optimization of the processing time may be required for certain tissue types, a program containing 20 cycles of 35,000 psi held for 20 seconds and atmospheric pressure held for 20 seconds may be a good starting point. 4. Remove the PULSE Tube(s) from the instrument and Vortex them for 10-20 seconds. 5. Pipette the entire contents of the PULSE Tube Into a 2mL centrifuge collection tube. To remove any remaining liquid, slowly and carefully push the ram into the barrel of each PULSE Tube as far as it can go using the special tool provided. Remove any remaining liquid extract with a pipette until all of the liquid is transferred from the PULSE Tube(s).

6. Centrifuge sample for 15 minutes at 8,000×g. After centrifugation, the sample will separate into 2 phases: an upper phase that contains lipids, and a lower organic hydrophilic phase that contains proteins.

A white residue may be noticeable under certain conditions at the interface between two liquid phases. In the case of adipose tissue samples, this interphase contains mainly blood vessels, collagenous extracellular matrix and other proteins of non-cellular origin. If the interphase is visibly large, however, it may be an indicator of insufficient extraction due to the large tissue-to-solvent ratio. Re-extraction of this material in the original PULSE Tube with a fresh aliquot of Reagent A will ensure complete dissolution of the cellular proteome. Resulting extracts may be pooled.

8. Transfer the lower phase into new sample tubes. If desired, store the original sample tubes con taining upper lipid phase for future lipid analy sis. 9. Lower phase extracts can be split into several aliquots at this stage. This provides a convenient way to aliquot the protein extract. Smaller aliquots also tend to evaporate at a considerably faster rate.

10. For solvent removal by evaporation, continue to step 11. For solvent removal by precipitation, skip to step 12. 11. Place lower phase aliquots into a vacuum evaporator. Evaporate contents until 510 µl of liquid remains in the centrifuge tube. Skip to step 13 WARNING: Avoid evaporation of the sample to dryness! Over dried material may be difficult to dissolve due to protein aggregation.

12. To each sample add a 3-fold excess (v:v) of Re agent C. Rapidly mix the samples. Increased sample turbidity should be immediately ob- served. Chill samples on ice for 10 minutes. 13. Centrifuge the precipitated samples for 15 minutes at 8,000 g. Aspirate or decant the super-natant. If necessary, spin tube again briefly and remove residual supernatant. 14. Reconstitute the pellets in a buffer/reagent suitable for downstream analysis. Protocol is com-pleted at this step.

Troubleshooting Guide Problem

Possible Cause

Remedy

Low protein yield Sample degradation (proteolysis) prior to extraction

Maintain samples frozen until they are inserted into the PULSE Tubes. Add reagent A and extract as soon as possible. Proteolysis should be abolished in Reagent A.

Sample degradation (proteolysis) in the reconstitution buffer

Maintain sufficient concentration of protease inhibitors in the reconstitution media. It is unlikely that sample-derived protease activity will be restored upon reconstitution.

Tissue sample amount is too small

Increase tissue sample size, if possible. Use smaller volume of reagent A and increase volume of Reagent B to maintain total volume of 1.4 mL per PULSE Tube.

Inefficient protein precipitation

Dry samples in vacuum evaporator.

Proteolysis

Protein below detection limit

Incomplete sample dissolution.

Formation of a visible white interface which does not disappear after centrifugation. Tissue appears not fully homogenized

Ram is pushed out of PULSE tube.

Excessive amount of tissue per PULSE Tube

Decrease the amount of tissue per PULSE Tube or split sample between several PULSE Tubes as required.

Tissue sample contains high amount of hydrophilic material

Re-extract with a fresh load of Reagent A

Sample contains highmelting lipids

Increase processing temperature

Total sample volume exceeds 1.4 ml

Use less Reagent A and/or B

Oil from sample is lubricating ram O-rings

Use clip to keep ram in place

Pulse tube is collapsed or has a pinhole Total sample volume is less than 1.2 ml

Use more Reagent A and/or B

Liquid phases do not form clean interface. One of the phases or both appear cloudy.

Phase separation is not well defined

Insufficient amount of sample-derived lipid to form a layer

Add 200 µl of Reagent B to the PULSE Tube before processing. Maintain constant total volume of 1.2mL - 1.4mL in the PULSE Tube.

Centrifugation time is insufficient to separate phases.

Vortex the sample tubes for 10 seconds. Centrifuge for 15 minutes at or below 8,000 g. Never exceed 8,000 g while spinning kit reagents in the supplied 2mL centrifuge tubes to avoid possible tube failure and sample loss

Very high water content in the sample

Aspirate the lower phase into a separate container. Add more Reagent A, vortex and centrifuge as described above.

Third clear phase is visible on the liquid interface

Multiple hydrophobic phases form

Decrease the amount of tissue per PULSE Tube or split sample between several PULSE Tubes as required. Re-extract with a fresh load of Reagent A Increase processing temperature

Solvent evaporation exceeds 4 hours.

Substantial lipid carry-over

Lipid layer forms during evaporation

Repeat aspiration of the lipid phase, if visible, and then continue evaporation. Precipitate sample with reagent C (see step 11)

Insufficient vacuum

None of the samples, including pure Reagent A, evaporate quickly

Check vacuum pump, connections, seals and a cold trap for leaks. If necessary, have the evaporator serviced.

Discovery Starts With Sample Preparation

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