Name: preparation of cell extracts by cryogrinding in an automated freezer mill
Uploaded: 2021-01-29T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill at JoVE.com

Research in the Gunjan lab is supported by funding from the National Institutes of Health, National Science Foundation and the Florida Department of Health. We thank undergraduate student John Parker for technical assistance.

1. Preparation of Yeast Popcorn
<ol>
	<li>Grow yeast cells in 0.5 L of YPD media to a density of 1 x 107 cells/mL. Count cells using a Coulter Counter or any other means.</li>
	<li>Centrifuge cells for 10 min at 2,400 g and 4 &#176;C.</li>
	<li>Wash each sample once with 500 mL of ice-cold deionized 18 mega Ohm Milli-Q water.</li>
	<li>Resuspend pellets in 15 mL of ice-cold Lysis Buffer [20 mM HEPES-KOH pH 7.5, 110 mM KCl, 0.1% Tween, 10% glycerol, with freshly added reducing agent 10 mM &#946;-mercaptoethan...

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A limitation of studying native proteins from yeast is the inefficient lysis of yeast cells due to&#160;their tough cell wall. Although several methods have been developed, the most consistent and efficient method in our hands is the cryogrinding of yeast cells flash frozen as popcorn. This method allows the reliable preparation of high-quality whole cell extracts from budding yeast compared to other lysis methods. The representative results demonstrated that cryogrinding is superior to a popular mechanical method for ye...

Yeast is a popular model organism for protein studies, as it is a simple eukaryotic organism with an abundance of genetic and biochemical tools available for researchers1. Because of their sturdy cell wall, one challenge that researchers face is in efficiently lysing the cells without damaging the cellular contents. Different methods are available for obtaining protein extracts through disruption of yeast cells which include enzymatic lysis (zymolyase)2,3, chemical lysis4, physical lysis by freeze-thaw5, pressure-based (French press)6,7, mechanical (glass beads, coffee grinder)8,9, sonication-based10 and cryogenic2,11. The efficiency of cell lysis and the protein yield can vary considerably depending on the technique employed, thus affecting the end result or suitability for the desired downstream application for the lysate. When studying proteins that are unstable, have fleeting posttranslational modifications, or are temperature sensitive, it is particularly important to use a method that will minimize sample loss or degradation during preparation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Extract preparation technique</td>
			<td>Details</td>
			<td>Advantages</td>
			<td>Disadvantages</td>
			<td>Downstream analysis</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>French press: High-pressure homogenizer (aka Microfluidizer) with enzymatic pretreatment using Zymolyase</td>
			<td>Zymolyase-20T, a Microfluidizer high-pressure homogenizer. The disruptor consists of an air-driven, high-pressure pump (ratio 1:250; required air pressure 0.6-l MPa) and a special disruption chamber with an additional back pressure unit. A minimum sample size of 20 mL is required for processing.</td>
			<td>Final total disruption obtained using the combined protocol approached 100 % with 4 passes at a pressure of 95 MPa, as compared to only 32 % disruption with 4 passes at 95 MPa using only homogenization without the Zymolyase.</td>
			<td>Not appropriate for small scale applications. The enzymes can get expensive for large scale preparations.</td>
			<td>Protein purification</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Bead beater: Zymolyase treated cells lysed with glass beads in a fastprep instrument</td>
			<td>Roughly an equal volume of cold, dry, acid-washed 0.5 mm glass beads is added to a given volume of cell pellet in lysis buffer and the cells are disrupted by vigorous manual agitation.</td>
			<td>It is particularly useful when making extracts from many different small yeast cultures for assaying purposes rather than for protein purification.</td>
			<td>During the glass bead procedure, proteins are treated harshly causing extensive foaming leading to protein denaturation. The amount of cell breakage varies, while proteolysis as well as modification of the proteins may result from heating of the extract above 4&#176;C during the mechanical breakage.</td>
			<td>Mostly DNA &#38; RNA analyses, but also protein analysis by denaturing gel electropheoresis, either with or without Western blotting.</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Zymolyase treatment&#160;&#160; followed by lysis using a combination of osmotic shock and Dounce homogenization</td>
			<td>After enzymatic digestion of cell walls, spheroplasts are lysed with 15 to 20 strokes of a tight-fitting pestle (clearance 1 to 3 &#181;m) in a Dounce homogenizer.</td>
			<td>Advantageous to use protease-deficient strains such as BJ926 or EJ101. This is the gentlest way to break yeast cells and hence it is most suitable for preparing extracts that can carry out complex enzymatic functions (e.g., translation, transcription, DNA replication) and in which the integrity of macromolecular structures (e.g., ribosomes, splicesomes) has to be maintained. It is also useful for isolating intact nuclei that can be used for chromatin studies (Bloom and Carbon, 1982) or for nuclear protein extracts (Lue and Kornberg, 1987).</td>
			<td>The major disadvantages of the spheroplast lysis procedure are that it is relatively tedious and expensive, especially for large-scale preparations (&#62;10 liters), and the long incubation periods can lead to proteolysis or protein modification. &#160;For chromatin preparations, they seem to be of varying or lower quality than those produced by the differential centrifugation (based on nucleosome ladder integrity).</td>
			<td>Isolating intact nuclei for chromatin studies, extracts that can carry out complex enzymatic functions, extracts requiring the integrity of 
			macromolecular structures, nuclear protein extracts.</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Cell Disruption of flash frozen cells by grinding in Liquid Nitrogen using a mortar/pestle or a blender</td>
			<td>Cells are frozen immediately in liquid nitrogen and then lysed by grinding manually in a mortar using a pestle, or using a Waring blender in the presence of liquid nitrogen.</td>
			<td>The protocol is quick and easy. It can accommodate varying amounts of yeast cells including very large cultures. Its main advantage is that cells are taken immediately from the actively growing state into liquid nitrogen (&#8722;196&#176;C), decreasing degradative enzyme activities such as proteases and nucleases as well as activities that modify proteins (e.g., phosphatases and kinases). It is particularly suited for making whole-cell extracts from a single yeast culture for large-scale protein purification.&#160;</td>
			<td>A bit messy and potentially dangerous to the careless investigator. Small samples (i.e., 10- to 100-ml yeast cultures) are not easily processed because there is not enough mass of frozen cell clumps to fracture effectively in the blender. It is time-consuming to process individual samples and to clean the equipment between uses.</td>
			<td>Whole-cell extracts from a single yeast culture for large-scale protein purification.</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Autolysis, Bead mill</td>
			<td>pH 5.0, 50 &#176;C, 24 h, 200 rpm / &#216; 0.5 mm, 5 &#215; 3 min/3 min</td>
			<td>Quick and efficient lysis, especially for small scale extract preparation</td>
			<td>Heat generation leads to denaturation and degradation of macromolecules. Bead beating equipment required.</td>
			<td>Small scale analyses.</td>
			<td rowspan="2">10</td>
		</tr>
		<tr>
			<td>Autolysis, Sonication</td>
			<td>pH 5.0, 50 &#176;C, 24 h, 200 rpm, 4 &#215; 5 min/2 min, pulser 80%, power 80%</td>
			<td>Sonication equipment is usually available in most institutions.</td>
			<td>Heat generation leads to denaturation and degradation of macromolecules. Sonication equipment required. Slow lysis can take more than 24 hours.</td>
			<td>Yeast cell wall preparations.</td>
		</tr>
		<tr>
			<td>Boiling and freeze-thaw process</td>
			<td>No specialized equipment needed other than a standard freezer and a heating block or hot water bath.</td>
			<td>Efficient, reproducible, simple and inexpensive.</td>
			<td>Heat generation leads to denaturation and degradation of macromolecules.</td>
			<td>DNA analyses by PCR.</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Comparison of methods available for the preparation of yeast extracts.
Cryogrinding (aka cryogenic grinding/cryogenic milling) is commonly employed to retrieve nucleic acids, proteins or chemicals from temperature sensitive samples in a reliable manner for quantitative or qualitative analyses. It has been used successfully for multiple applications in diverse fields including biotechnology, toxicology, forensic science12,13, environmental science, plant biology14 and food science. Isolation of intact biological macromolecules is usually critically dependent on the temperature. Extremely low temperatures ensure that the proteases and nucleases stay inactive, resulting in a reliable isolation of intact proteins, nucleic acids and other macromolecules for subsequent analyses. Indeed, a freezer mill typically maintains a sample temperature of -196 &#176;C (the boiling point of LN2), thus minimizing DNA/RNA or protein denaturation and degradation.
The freezer mill employs an electromagnetic grinding chamber that rapidly moves a solid metal bar or cylinder back and forth within a vial containing the sample to be pulverized between stainless steel end plugs. The instrument creates and rapidly reverses a magnetic field within the grinding chamber. As the magnetic field shifts back and forth, the magnet crushes the sample against the plugs thus achieving the &#39;cryogrinding&#39; and the pulverization of the popcorn. The freezer mill replaces the mortar and pestle and allows the sequential processing of multiple samples (or up to 4 smaller samples simultaneously) with high reproducibility and avoids the user-to-user variability associated with manual grinding. Once the samples are processed, the cell extracts can be used for a variety of downstream applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>50 mL polycarbonate tubes with screw caps</td>
			<td>Beckman</td>
			<td>357002</td>
			<td>Centrifuge tubes</td>
		</tr>
		<tr>
			<td>BD Bacto Peptone</td>
			<td>BD Biosiences</td>
			<td>211677</td>
			<td>Yeast YPD media component</td>
		</tr>
		<tr>
			<td>BD Bacto Yeast Extract</td>
			<td>BD Biosiences</td>
			<td>212750</td>
			<td>Yeast YPD media component</td>
		</tr>
		<tr>
			<td>Beckman Avanti centrifuge</td>
			<td>Beckman</td>
			<td>B38624</td>
			<td>High speed centrifuge</td>
		</tr>
		<tr>
			<td>Beckman JLA-9.1000</td>
			<td>Beckman</td>
			<td>366754</td>
			<td>Rotor</td>
		</tr>
		<tr>
			<td>D-(+)-Dextrose Anhydrous</td>
			<td>MP Biomedicals</td>
			<td>901521</td>
			<td>Yeast YPD media component</td>
		</tr>
		<tr>
			<td>Eppendorf A-4-44</td>
			<td>Eppendorf</td>
			<td>22637461</td>
			<td>Swinging bucket rotor</td>
		</tr>
		<tr>
			<td>Eppendorf refrigerated centrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td>22625101</td>
			<td>Refrigerated centrifuge</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>SIGMA-ALDRICH</td>
			<td>G5150-1GA</td>
			<td>Volume excluder and cryoprotectant</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>FisherBiotech</td>
			<td>BP310-100</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>HIS6 antibody</td>
			<td>Novagen</td>
			<td>70796</td>
			<td>Antibody for HIS tag</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>SIGMA-ALDRICH</td>
			<td>P9541-1KG</td>
			<td>Salt for maintaining ionic strength</td>
		</tr>
		<tr>
			<td>MG-132</td>
			<td>CALBIOCHEM</td>
			<td>474790</td>
			<td>Proteasome Inhibitor</td>
		</tr>
		<tr>
			<td>Phosphatase inhibitor cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>A32957</td>
			<td>Phosphatase inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>SIGMA</td>
			<td>P7170-1L</td>
			<td>Protein Stain</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>A32963</td>
			<td>Protease inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Rotor JLA 25.500</td>
			<td>Beckman</td>
			<td>JLA 25.500</td>
			<td>Rotor</td>
		</tr>
		<tr>
			<td>Sodium Butyrate</td>
			<td>EM Science</td>
			<td>BX2165-1</td>
			<td>Histone Deacetylase Inhibitor</td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>S6521</td>
			<td>Phosphatase Inhibitor</td>
		</tr>
		<tr>
			<td>Sodium Vanadate</td>
			<td>MP Biomedicals</td>
			<td>159664</td>
			<td>Phosphatase Inhibitor</td>
		</tr>
		<tr>
			<td>Sodium &#946;-glycerophosphate</td>
			<td>Alfa Aesar</td>
			<td>13408-09-8</td>
			<td>Phosphatase Inhibitor</td>
		</tr>
		<tr>
			<td>Spex Certiprep 6850 freezer mill</td>
			<td>SPEX Sample Prep</td>
			<td>6850</td>
			<td>Freezer Mill</td>
		</tr>
		<tr>
			<td>TALON Metal Affinity Resin</td>
			<td>BD Biosiences</td>
			<td>635502</td>
			<td>For pulling down HIS tagged proteins</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>VWR International</td>
			<td>VW1521-07</td>
			<td>Non-ionic detergent</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>AMRESCO</td>
			<td>M131-250ML</td>
			<td>Reducing agent</td>
		</tr>
	</tbody>
</table>

preparation of cell extracts by cryogrinding in an automated freezer mill

The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae has made it a pre-eminent genetic model organism. However, since efficient protein isolation depends upon optimal disruption of cells, the use of yeast for biochemical analysis of cellular proteins is hampered by its cell wall which is expensive to digest enzymatically (using lyticase or zymolyase), and difficult to disrupt mechanically (using a traditional bead beater, a French press or a coffee grinder) without causing heating of samples, which in turn causes protein denaturation and degradation. Although manual grinding of yeast cells under liquid nitrogen (LN2) using a mortar and pestle avoids overheating of samples, it is labor intensive and subject to variability in cell lysis between operators. For many years, we have been successfully preparing high quality yeast extracts using cryogrinding of cells in an automated freezer mill. The temperature of -196 &#176;C achieved with the use of LN2 protects the biological material from degradation by proteases and nucleases, allowing the retrieval of intact proteins, nucleic acids and other macromolecules. Here we describe this technique in detail for budding yeast cells which involves first freezing a suspension of cells in a lysis buffer through its dropwise addition into LN2 to generate frozen droplets of cells known as "popcorn". This popcorn is then pulverized under LN2 in a freezer mill to generate a frozen "powdered" extract which is thawed slowly and clarified by centrifugation to remove insoluble debris. The resulting extracts are ready for downstream applications, such as protein or nucleic acid purification, proteomic analyses, or co-immunoprecipitation studies. This technique is widely applicable for cell extract preparation from a variety of microorganisms, plant and animal tissues, marine specimens including corals, as well as isolating DNA/RNA from forensic and permafrost fossil specimens.

We compared two different methods for yeast cell lysis, namely glass bead milling at 4 &#176;C and an automated cryogrinding method at -196 &#176;C, to assess the relative recovery proteins in the cell extracts prepared with both methods. For this study, we chose to use a budding yeast strain YAG 1177 (MAT a lys2-810 leu2-3,-112 ura3-52 his3-&#916;200 trp1-1[am] ubi1-&#916;1::TRP1 ubi2-&#916;2::ura3 ubi3-&#916;ub-2 ubi4-&#916;2::LEU2 [pUB39] [pUB221])16 carrying a ...

Watch this Scientific Journal Video about Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill at JoVE.com

Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill

We describe a reliable method for the preparation of whole cell extracts from yeast or other cells using a cryogenic freezer mill that minimizes degradation and denaturation of proteins. The cell extracts are suitable for purification of functional protein complexes, proteomic analyses, co-immunoprecipitation studies and detection of labile protein modifications.

The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae ...

Yeast is popular model organism, not just for genetic studies but also for biochemical studies of proteins and other macromolecules extracted from wild type and mutant yeast strains. However, due to their tough cell walls, a major challenge that researchers face is this efficient lysis of yeast cells without damaging the cellular content. The isolation of intact biological macromolecules is usually critical, dependent on temperature.Preparing the extracts at low temperature ensures that the intracellular proteases and nucleases stay inactive, resulting in reliable isolation of intact protein, nucleic acids, and other macromolecules for sufficient lysis. Various methods are available for obtaining yeast extracts through enzymatic, chemical and physical lysis. However enzymatic lysis is expensive due to the requirement of purified enzymes to digest the cell wall, thus limiting the use of smaller number of cells.Further the enzymatic reactions has to be monitored closely to prevent over digestion and premature lysis of the cells. Chemical methods using strong denaturing agents, though effective, result in the denaturation of proteins and as such, are not suitable for the isolation of protein complexes or functional enzymes. Physical methods, such as lysis under high pressure in a French Press can be carried out in the cold at four Celsius but that is not cold enough to prevent degradation and denaturation of all native proteins.Other popular physical methods are the use of a mortar and pestle, blender, or coffee grinder to grind yeast cells resuspended in a lysis buffer and frozen as droplets or grinding and mixture of glass beads and yeast in a fast prep bead beater mill or a lysis by sonication. However, manual grinding is labor intensive and the resulting protein yield can vary considerably depending on the techniques employed while lysis by sonication or grinding in a blender, coffee grinder, or bead beater mill generates a substantial amount of heat, which results in a protein denaturation and degradation. Therefore, to obtain yeast cell lysis with proteins in their native state with minimal denaturation and degradation for applications like purifications of functional protein complexes, biochemical assays or detection of fleeting post-translational modifications, it is essential to use an easily scalable lysis method that will minimize heat generation while allowing for efficient lysis regardless of the quantity of cells.Here we describe a method that involves the use of a cryo freezer mill for the lysis of cells in liquid nitrogen at minus 196 Celsius to generate frozen cell extracts thereby allowing the recovery of intact functional macromolecules, such as proteins or DNA and RNA, at a very low temperatures which minimizes denaturation and degradation. This method can be readily adapted for use with any type of cell or tissue sample from any species, including forensic and even ancient samples recovered from archeological sites or found frozen in permafrost. The freezer mill uses a superconducting electromagnetic grinding chamber that rapidly moves a solid metal bar back and forth within a polycarbonate file containing the sample to be pulverized between stainless steel end plugs.This physical cell lysis method allows for a more efficient lysis and reproducibly results in a better quality extract. Yeast cells were grown at a concentration of 10 million cells per mil. These yeast cells were placed pre chilled centrifuge bottle and were spun for 10 minutes at 2, 400 G.Once the spin is complete, decant the supernatant without disturbing the pelleted cells.This pellet was resuspended in a small amount of ice cold water, about half the initial volume of the culture to wash the cells. The resuspended cells were again spun down for another 10 minutes at 2, 400 G.Once the next spin is complete, we, again, decant all the supernatant without disturbing the pellet. This pellet will then be resuspended and 15 ML of ice cold lysis buffer.Ensure that the pellet is completely resuspended. The resuspended cells should remain on ice in a pre-marked tube until ready for the next step. Always wear personal protective equipment when handling liquid nitrogen, such as safety glasses, hand protection, and lab coats.To maximize our dexterity, we often wear a pair of white thermal gloves sandwiched between two pairs of nitrile gloves. Change the outer and nitrile gloves frequently, as they rip easily after being exposed to liquid nitrogen. In our lab, we transfer an aliquot of liquid nitrogen into a small five liter liquid nitrogen container.Liquid nitrogen is then poured into a 50 ML tube that has been pre chilled on dry ice. Once the 50 ML tube is full of liquid nitrogen, we add the yeast extract slowly, in a drop wise manner to the tube in 1.5 milliliter increments. To minimize the formation of large popcorn pellets, the yeast suspension is added in a circular motion.To further prevent the formation of large popcorn pellets, we use large forceps to ensure all pellets are between 0.3 and 0.5 centimeters in diameter. Because liquid nitrogen is always evaporating, be sure to refill the tube with liquid nitrogen before adding each 1.5 mil aliquot of yeast suspension. Once all of the yeast suspension has been made into popcorn allow the tube with popcorn to sit without the lid for two to three minutes to let all of the residual liquid nitrogen to evaporate.To be sure that all of the liquid nitrogen has evaporated, close the lid nearly completely and shake gently. If there is a hissing noise, it indicates that the liquid nitrogen has not yet fully evaporated. Open the lid for another minute or so to allow the remaining liquid nitrogen to evaporate before closing the lid tightly.Residual liquid in the tube may cause an explosion if the lid is closed prematurely. This yeast popcorn can be stored almost indefinitely in minus 80 degrees C.For at least five samples, ensure that you have 30 to 35 liters of liquid nitrogen available. Fill the freezer mill to the full line and close the lid to allow the freezer mill to pre chill for a few minutes.Again, always remember to wear personal protective gear when dealing with liquid nitrogen. The polycarbonate grinding vials, stainless steel end plugs, and impactor bar must be pre chilled with liquid nitrogen. Keep these components submerged until the liquid nitrogen stops bubbling.Remember to keep all popcorn samples on dry ice until the entire grinding process is complete. Pull out all the liquid nitrogen from the grinding vials and transfer your popcorn into the vial. Don't forget to place a magnetic impactor bar in the grinding vial as well.Seal the grinding vial by carefully placing the stainless steel plug evenly on the end of the grinding vial. We bang each end of the grinding vial on a wooden breadboard with a vibration absorbing rubber backing to ensure that the end plugs are placed correctly and are sealing the grinding vials tightly. It is important to secure the end plugs to ensure that they will not come loose and allow the samples to spill in the freezer mill during the grinding process.Place a grinding vial in the freezer mill grinding chamber and lock in place. Close the freezer mill lid and grind the sample for three cycles at two minutes per cycle, at a crushing rate of 14. After the grinding cycles are complete unlock the vial with the frozen powder cell lysate.To prevent the lysate from thawing out, work quickly and carefully to unscrew one of the end plugs using the opening tool. Once the vial is open, remove the impactor bar and quickly transfer the frozen powder extract on a pre chilled plastic weighing dish. Recover as much of the frozen extract as possible by banging the vial on the wooden breadboard.Once all the powder is removed from the vial, quickly transfer all of the powder extracts into a pre-labeled 15 mil tube kept on dry ice. Although it is best to proceed with thawing and using the extracts right away to minimize protein degradation, the frozen powder lysate can be stored overnight at minus 80, if needed. Thaw out the powder lysate slowly in an ice slurry that is continuously circulated using a magnetic stir bar in an ice bucket.To facilitate even thawing, remove ice that develops on the outside of the tubes frequently, about every five minutes or so. Once the extracts begin to thaw at 1X protease inhibitor cocktail and 10 micromolar proteasome inhibitor MG132 to prevent protein degradation. Once the samples are completely thawed, which can take over an hour, spin down the samples at 3, 220 G for 20 minutes at four Celsius degree.This spin will remove most of the cell debris from the lysate. When the spin is complete transfer the supernatant into polycarbonate bottles that have been pre chilled on ice and discard the pellet. Centrifuge the supernate and samples at 16, 000 G at four Celsius degree for 20 minutes to clarify the extract.After the spin is complete, recover only the clear supernatant carefully from the middle of the liquid column without disturbing the cloudy layer containing the piece on top or the pelleted debris at the bottom of the centrifuge tube. Transfer the clear lysate into a fresh pre chilled 15 Mil tube. The remaining cloudy liquid can be recentrifuged for five minutes at the same speed to allow the recovery of some more of the wholesale lysate.The extracts are now ready for use in experiments, such as purification of protein complexes and immunoprecipitation. We compared two different methods of yeast cell lysis, namely glass bead milling at four Celsius and an automated cryo grinding method at minus 196 Celsius to assess the relative recovered proteins and cell extracts prepared with both methods. From this study we chose to use a budding yeast strain carrying a high copy plasmid expressing a tandem HIS-MYC tagged ubiquitin.So we can assess the affinity of recovery of tagged polyubiquitinated proteins from wholesale extracts, following affinity purification using TALON cobalt HIS tag affinity beads followed by Western blotting with a monoclonal HIS tag antibody. Polyubiquitinated proteins that are normally very short-lived due to their rapid degradation by the ubiquitin proteasome machinery and thus offer a very good way to compare the quality of wholesale extracts prepared by different methods. As we see by the right half of the Ponceau stained membrane, we recovered a higher total protein yield in the wholesale extracts prepared by the cryo freezer mill protocol as judged by the more intensive Ponceau staining throughout the wholesale extract lane.This is especially clear in the lower and upper parts of the freezer mill whole extracts lanes. Additionally, as shown by the right half of the HIS tag Western blot, we observed a better recovery of HIS-MYC tag ubiquitinated protein followed by pull down using Talon beads from the wholesale extracts prepared by cryogenic grinding. We conclude that the cryogenic freezer mill is superior to other methods for the preparation of cell extracts, especially when functional macromolecules are required for downstream application.

preparation-cell-extracts-cryogrinding-an-automated-freezer

Research

JoVE Journal

Biology

Title Cell Encapsulation by Droplets

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...