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;-mercaptoethanol, protease inhibitor cocktail, 10 &#956;M proteasome inhibitor MG-132, 1 mM deacetylase inhibitor sodium butyrate and phosphatase inhibitors (1 mM sodium vanadate, 50 mM sodium fluoride, 50 mM sodium &#946;-glycerophosphate)]. See the&#160;Table of Materials&#160;for additional details. Keep resuspended cells on ice until the samples are ready for the next step. 
	NOTE: A wide variety of lysis buffers containing a number of non-ionic detergents can be used with the samples and specific inhibitors included in it based on the downstream application. Inclusion of protease and proteasomal inhibitors are crucial for preventing protein degradation, especially once the extracts are thawed.</li>
	<li>Make snap frozen yeast popcorn by slowly adding the cell suspension one drop at a time using a pre-chilled serological pipette into one or more 50 mL centrifuge tubes kept on dry ice and filled with LN2 until just below the rim. Top up liquid nitrogen in the tube frequently to compensate for its loss due to rapid evaporation. Work in a well-ventilated area to avoid the hazards associated with nitrogen asphyxiation.
	<ol>
		<li>Instead of a 50 mL tube, use multiple 50 mL tubes tied together (or any container with a large mouth, such as a large plastic centrifuge bottle that can tolerate cryogenic temperatures) for pop-corn preparation (Figure 1A). The larger the container and its opening, the easier is the popcorn preparation as this prevents clumping of the popcorn to form large aggregates. A size range of 0.3-0.5 cm for the popcorn is ideal for achieving proper grinding/pulverization using the freezer mill (Figure 1B).</li>
		<li>Ensure that the LN2 has evaporated completely from the tubes containing the popcorn prior to storing them at -80 &#176;C. It is possible to stop after preparing the yeast popcorn as they are stable for several years when maintained at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61164/61164fig01.jpg" /> 
Figure 1:&#160;Yeast popcorn preparation.&#160;(A)&#160;Yeast &#34;popcorn&#34; is made by the dropwise freezing of the cell suspension in LN2. We use one to three 50 mL tubes held together with a rubber band and placed in an ice bucket filled with dry ice. The tubes are filled with LN2&#160;until just below their rims and are topped up with liquid nitrogen frequently to keep them nearly full until all the cell suspension had been made into popcorn (B)&#160;The size of the popcorn is an important determinant of optimal grinding efficiency. The size range of the popcorn should be between 0.3 and 0.5 cm in diameter.&#160;<a href="https://www.jove.com/files/ftp_upload/61164/61164fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Cryogrinding
<ol>
	<li>To start, ensure that there is enough LN2 available to chill the freezer mill, grinding vials, supplies and to operate the machine for all samples. For less than five samples, 30-35 L of LN2 should suffice. Fill the freezer mill chamber with LN2 up to the fill line. 
	WARNING: All steps involving LN2 should be performed in a well-ventilated area and the freezer mill itself should be located in such an area to avoid risk of asphyxiation. Wear personal protective equipment including proper footwear, lab coat, safety glasses, a base layer of nitrile gloves, then thermal gloves, followed by another pair of nitrile gloves. Use extreme caution when handling LN2.</li>
	<li>Close the freezer mill lid slowly, avoiding the splash of LN2 and allow a few min for the machine to cool down. It may be necessary to refill the chamber with LN2 to compensate for the loss due to evaporation before proceeding to the next step. 
	NOTE: Refill LN2 as needed only up to the fill line. Do not overfill the chamber as this can be both dangerous and detrimental to the machine. Automated refill systems are available for freezer mill that can replenish the evaporating LN2 as needed directly from a storage tank connected to the freezer mill.</li>
	<li>Pre-chill the large grinding vials and the magnetic impactor bar by dunking them in LN2 kept in a separate small dewar. Decant all the LN2 when the LN2 stops bubbling. Then add the sample/yeast popcorn to the grinding vial and seal it tightly with the two stainless steel end plugs.
	<ol>
		<li>Do not fill more than one-third of the grinding vial with the sample as that can reduce the efficiency of grinding. Instead, larger samples can be processed by dividing them into two or more vials and grinding them sequentially. Smaller grinding vials can be used for smaller samples (up to 3 mL).</li>
	</ol>
	</li>
	<li>Place the grinding vial in the freezer mill chamber and lock it in place. Close the lid.</li>
	<li>For budding yeast, grind the samples for a total of three cycles for 2 min/cycle (with 2 min break for cooling between cycles) at a crushing rate of 14. When the machine stops after completing the cycles, open the freezer mill lid slowly and carefully unlock the vial with the powdered frozen cell lysate and remove it from the freezer mill. If multiple small vials are used, work quickly to remove one vial at a time and place them in dry ice. 
	NOTE: There is usually no need to adjust the grinding parameters when using vials of different sizes with the same type of sample. However, the grinding parameters such as the number of cycles and the grinding rate need to be empirically determined for different sample types, depending on the ease with which they lyse. Most mammalian cells and soft tissues will lyse in 1-2 cycles at a crushing rate of 10. Harder to lyse samples such as bacteria, yeast, fly larvae and adult fruit flies require 3-6 cycles at the maximum rate, while hard tissues such as bone, teeth, etc. may require up to 10 cycles. A small aliquot of the thawed sample can be viewed under the microscope before and after grinding to count the number of intact, unlysed cells to determine the lysis efficiency. An excellent analysis of the impact of cryogrinding parameters on the release of proteins and DNA from budding yeast has been published previously15.</li>
	<li>Working quickly so as not to allow the sample to thaw out, carefully unscrew one of the end pieces (pick the one that appears to have become somewhat loose during the grinding) using the opening tool. Then, use a pair of long forceps (that are pre-chilled in a bucket with dry ice) to remove the impactor bar. Collect the pulverized sample by inverting and tapping the grinding vial onto a polystyrene weighing dish pre-chilled with LN2 and kept on dry ice.</li>
	<li>Once all of the powdered frozen cell lysate has been recovered from the grinding vial, pour the powdered lysate from the weighing dish back into the 50 mL tube and proceed immediately to the slow thawing step described next for best results. Alternatively, store the frozen powdered lysate overnight at -80 &#176;C, although this may result in some degradation.</li>
	<li>Prepare an ice bucket with a 50% slurry of ice and water and place it on a stir plate with a magnetic stirrer. Submerge a wire rack or another suitable rack in the ice slurry to hold the samples.
	<ol>
		<li>Then, slowly thaw the samples on an ice slurry bath with constant agitation of the slurry using a magnetic stirrer. Add more ice to replace the melting ice (some water may need to be discarded to prevent the ice bucket from overflowing). Since several inhibitors have short half lives in aqueous buffers, add additional protease inhibitor cocktail (see Table of Materials) and 10 &#956;M proteasome inhibitor MG-132 to the lysate once the samples start thawing (after approximately 30 min).</li>
		<li>Remove ice formed on the outside of the tubes every 5 min to expedite the thawing process. Note that rapid thawing of the samples at room temperature or higher can lead to significant degradation.</li>
	</ol>
	</li>
	<li>After the samples have thawed completely (it may take well over an hour depending on the amount of sample), centrifuge the lysate at ~3,220 g for 20 min in a refrigerated tabletop centrifuge at 4 &#176;C to remove the bulk of the cell debris from the lysate.</li>
	<li>Transfer the supernatant to 50 mL polycarbonate centrifuge tubes that have been pre-chilled on ice and centrifuge the samples at 16,000 g for 20 min at 4 &#176;C (see Table of Materials).</li>
	<li>Transfer only the clear supernatant consisting of the clarified whole cell extract from the center of the liquid column in the tube very slowly and carefully, without disturbing the cloudy lipid layer (Figure 2), into a chilled 15 mL tube using pre-chilled serological pipettes. Avoid removing all of the lysate in the centrifuge tube to prevent carryover of lipids and debris. The leftover lysate in the centrifuge tube can be centrifuged multiple times to recover small amounts of extract after each spin. 
	&#8203;NOTE: Lipids along with any denatured proteins would appear cloudy/milky and are located at the top of the liquid/air interface, and occasionally above the pellet itself that consists of insoluble cellular debris (Figure 2).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61164/61164fig02.jpg" /> 
Figure 2:&#160;Extract layers in a centrifuge tube.&#160;The major visible features of the whole cell extract in a tube following centrifugation at 16,000 g for 20 min are indicated. The relative abundance of each feature depends on the sample type, the growth phase of the cells (exponential versus stationary), the amount of lysis buffer used to resuspend cells and the lysis efficiency.&#160;<a href="https://www.jove.com/files/ftp_upload/61164/61164fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="12">
	<li>Centrifuge any remaining lysate for 5 min to recover more of the extract. Repeat this 5 min centrifugation multiple times if needed to recover as much of the clear extract as possible. Discard the cloudy lysate containing lipids close to the bottom of the tube near the pellet.</li>
	<li>Use these extracts for downstream applications such as protein complex purification, immunoprecipitation and proteomic analysis.</li>
</ol>

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The authors have nothing to disclose.

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 ...

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7.9K Views.  Florida State University College of Medicine. 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.

Video: Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill

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 ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

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.

Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill

Summary

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Title Cell Encapsulation by Droplets

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