Name: Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting (Video) | JoVE Journal
Uploaded: 2014-04-10T15:30:00.000+00:00
Duration: 10 min 51 s
Description: Learn a consensus brain protein extraction protocol for proteomics using human and murine tissue with 2D-DIGE and mini 2DE immunoblotting. This method enables reliable identification of post-translational modifications.

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5 minutes

This work was supported by Inserm, University of Lille 2, MEDIALZ, LabEx (excellence laboratory, program invest for the future) and DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer's disease). F-J.F-G is currently a recipient fellowship of ANR (French National Research Agency/ NeuroSplice de Tau Projet ANR- 2010-BLAN-1114 01), but this work was also under the support of a grant from the JCCM (Spain).

1. Homogenization and Total Protein Extraction from Human and Mouse Brain Tissue
<ol>
	<li>Brain tissue homogenization. Homogenize the brain tissue 10% (w/v) in 8 M urea, 2 M thiourea and 1% w/v SDS buffer (UTS) using a potter glass (for human samples) or Teflon homogenizer (for mice samples). Sonicate at 60 Hz 30 pulses with a ultrasound generator applying 0.5 sec for total tissue disintegration13,14.
	<ol>
		<li>Determine protein concentration with Bradford assay, using BSA as standard. This UTS buffer extraction is totally compatible with one dimension SDS-PAGE as long as lysates are not over-heated to avoid carbamylation15.</li>
		<li>Add 1% (w/v) of Amberlite and incubate with gentle mixing during 10 min at room temperature. Thereafter filter onto 0.22 &#181;m syringe filters and finally add the SDS to the urea/thiourea solution. This step reduces the amount of uric acid and isocyanic acid, which are in equilibrium with urea in solution and facilitate its degradation.</li>
	</ol>
	</li>
	<li>Chloroform/methanol protein precipitation. Precipitate between 100-150 &#181;g of protein for 11 cm IPG Strips and 1-1.5 mg for 18 cm ones (independently on the pH range selected to perform the IEF).
	<ol>
		<li>Perform all the steps on ice or at 4 &#176;C. Cool chloroform and methanol at 4 &#176;C for 30 min before starting the precipitation procedure.</li>
		<li>Add three volumes of methanol and one volume of chloroform to one volume of UTS lysate. Shake manually the mixture and add three volumes of cold water. Vortex during 1 min and pellet the proteins by centrifugation at 12,000 x g for 30 min at 4 &#176;C.</li>
		<li>Discard the supernatant using a Pasteur pipette and add three volumes of cold MeOH. Vortex again and centrifuge at 12,000 x g during 30 min at 4 &#176;C. Finally discard supernatant and dry the pellet under N2.</li>
	</ol>
	</li>
	<li>Preparation of proteins for 2D analysis. Resuspend the protein-dried pellet in 2D-buffer (8 M urea, 2 M thiourea supplemented with 4% CHAPS). Wash the solution with Amberlite as indicated above and try to keep the proportion of 500 &#181;g of protein per 200 &#181;l of 2D buffer for 2D-DIGE. Note: 2D buffer can be stored at -80 &#176;C or kept at 4 &#176;C during one week (to prevent urea degradation).
	<ol>
		<li>Sonicate and store the samples at -80 &#176;C until carrying out the following step. Noteworthy, this protocol can be used after formic acid solubilization of protein aggregates10. Briefly, evaporate formic acid under N2 and resuspend the pellet as described.</li>
	</ol>
	</li>
</ol>
2. 2D-DIGE
<ol>
	<li>Samples test quality. Dose samples again using Bradford method. Dilute 15 &#181;g of proteins in LDS sample buffer, heat at 37 &#176;C15 and load onto 4-12% polyacrylamide gels.</li>
	<li>Coomassie staining. Stain polyacrylamide gel with 0.1% (w/v) Coomassie Blue G-250, 50% EtOH and 10% (v/v) acetic acid solution for at least 1 hr. Let background destain overnight in 7% acetic acid and 10% (v/v) ethanol solution.</li>
	<li>Pre-electrophoretic labeling of samples. Prior to performing cyanine dye labeling, verify the pH of the sample, which should be basic or even neutral since the N-hydroxysuccinimide substitution is more efficient at basic pH and given to be optimal at pH 8.6.
	<ol>
		<li>Label minimally the two samples of study with Cy3 or Cy5 fluorescent dyes with a ratio of 50 &#956;g protein/400 pmol dye and keep for 1 hr at 4 &#176;C in the darkness to facilitate the NHS ester reactive group of the cyanines with the unprotonated amine groups of lysines.</li>
		<li>Reduce sample variations making a cross-labeling with Cy3 and Cy5 dyes, avoiding a preferential coupling of one sample to a particular cyanine. Note that the cyanine-dye minimal labeling can be adapted for small amount of proteins with keeping the proportion of 1 &#181;g protein per 8 pmol cyanine-dye. If so, the mini-2DE system can be used instead of the large 2D gel system. This will enable the 2D-DIGE analysis for a small amount of brain tissue.</li>
		<li>Label a pool of both samples with the same amount of protein (50 &#181;g in total) with Cy2 fluorescent dye and used as internal standard. Use the same conditions as previously indicated.</li>
		<li>Complete to a total volume 350 &#956;l with 2D buffer containing 1.1% Destreak reagent, 1.2% of IPG buffer and traces of Bromophenol Blue the total 150 &#181;g of labeled proteins (50 &#181;g of Cy2, 50 &#181;g of Cy3 and 50 &#181;g of Cy2). Perform this step in quadruplicate using four independent strips. Moreover, prepare two additional non-labeled strips (one for each sample) with 350-450 &#181;g of protein for the preparative gels.</li>
		<li>Leave the six loaded strips covered with mineral oil overnight for passive rehydration.</li>
	</ol>
	</li>
	<li>Isoelectrofocusing. Carry out the IEF at 20 &#176;C. For a pH 3-11NL, 18 cm strip the protocol is: maximum current setting is 50 &#956;A/strip during 8 stages: 1) 150 V step for 1 hr, 2) 200 V step for 5 hr, 3) 500 V gradient for 2 hr, 4) 1,000 V gradient for 2 hr, 5) 2,000 V gradient for 2 hr, 6) 4,000 V gradient for 2 hr, 7) 8,000 V gradient for 2 hr and 8) for a total of 24,000 V&#183;hr step. After IEF, dispatch the excess of mineral oil using chromatography paper and store the strips at -20 &#176;C until the second dimension.</li>
	<li>IPG Strips equilibration. Equilibrate strips in 6 M urea, 2% SDS, 30% Glycerol, 50 mM Tris-HCl pH 8.6 buffer with 1% of DTT during 15 min and after with 4.7% of iodoacetamide in the same buffer but without DTT.</li>
	<li>Second dimension or SDS-PAGE electrophoresis. Make the six 12% SDS-PAGE gels (1.5 M Tris-HCl pH 8.6, 12% acrylamide/bisacrylamide, 0.1% (w/v) SDS, 0.072% (w/v) APS and 0.1% (v/v) TEMED) at the same time using a large 2D system, with four low-fluorescence glass plates for the strips with fluorescence samples and two other glass plates with 1.5 mm of thickness for preparative gels in order to excise the protein spots.
	<ol>
		<li>Load the six IPG Strips on the top of the gels and over layered with 0.5% (w/v) of agarose. Running buffer is composed of 25 mM Tris, 192 mM glycine and 0.1% (w/v) SDS. Carry out the migration at 2.5 W/gel overnight with a refrigeration system set to 4 &#176;C16,17.</li>
	</ol>
	</li>
	<li>Fluorescence images acquisition and analysis. Use a Typhoon 9400 scanner to obtain the fluorescence gels images (12 images for one experiment). Scan Cy2, Cy3 and Cy5 images for each gel at 488/520 nm, 532/580 nm and 630/670 nm excitation/emission wavelengths, respectively. Use informatic software to analyze the images. Data are representative of the changes of spot volume associated with the distribution of the signal across the four gels among the two samples studied.</li>
	<li>Coomassie staining for big gels. Fix the preparative gels in 50% (v/v) EtOH and 3% (v/v) orthophosphoric acid solution for at least 24 hr. Then after, wash twice with ultra pure water for 20 min. Perform coloration in 34% (v/v) MeOH, 17% (w/v) ammonium sulfate and 2% orthophosphoric acid for 1 hr before adding 0.1% (w/v) Coomassie Blue G-250. Keep the gels in coloration for at least 48 hr and decolorized in ultra pure water.
	<ol>
		<li>Spots for MS identification. Once fluorescence images are analyzed, the software provides a list of spots whose expression is statistically different. Manually excise these spots from the preparative gels. This procedure requires practice and some manual dexterity in order to avoid keratin contamination. Then samples can be kept in water at -20 &#176;C until sending to MS. Spot identification is perfectly compatible with Tandem mass spectrometry (MS/MS) and relevance of protein identities is judged according to the probability based Mowse score calculated with a p-value of 0.05 (p &#60; 0.05)18.</li>
	</ol>
	</li>
</ol>
3. Qualitative Mini-2DE Assay
<ol>
	<li>Resuspend maximum 100 &#181;g of protein sample in 2D buffer until a final volume of 200 &#181;l for a 11 cm IPG strip. In case of overexpression system or purified protein, the starting amount can be lowered to 20 &#181;g.</li>
	<li>Add 1.1% (v/v) Destreak Reagent, 0.55% (v/v) IPG buffer and traces of Bromophenol Blue to a final volume of 200 &#181;l. Then, vortex, make a fast spin and load into an IPG strip for passive rehydration (to bring the strip to its original thickness) overnight covered with mineral oil. Note: the proportion of IPG buffer is the same for all the interval of pH desired (pH 3-11, 4-7, 7-11, etc), but its composition varies in function of the pH range selected.</li>
	<li>Isoelectrofocalisation. Perform IEF at 20 &#176;C. The duration of the program depends on the pH interval selected. Noteworthy, parameters such as electroendosmosis may disturb the IEF profile and in these cases, optimization in electrofocalisation time is required19. Note: it is not necessarily a longer IEF that provides better results but shortening the time of IEF can also improve the resolution. After IEF, IPG strips can be stored at -20 &#176;C for months.</li>
	<li>IPG strips equilibration. Equilibrate the strips in a buffer containing 25 mM Tris-HCl pH 6.8, 20 mM DTT, 10% glycerol, 5% SDS, 0.05% Bromophenol Blue. Make three equilibration bathes for 15 min with changing the buffer solution between each bath.</li>
	<li>SDS-PAGE. Layer the IPG strip onto a precast gel (IPG+1 well, 11 cm IPG strip) with a percentage of polyacrylamide chosen depending on the MW of the protein of interest. Then, blot the gel onto a nitrocellulose/PVDF membrane at 100 mV during 33 min.</li>
	<li>Incubate overnight with the required antibody and visualize by peroxidase activity. Perform the alignments of immunoblots using unrelated polypeptides revealed by the secondary antibody. Reach a semi-quantitative analysis by densitometry of the volume and intensity for the traces/ spots with ImageJ software.</li>
</ol>

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<li>Leboucher, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detrimental+effects+of+diet-induced+obesity+on+tau+pathology+are+independent+of+insulin+resistance+in+tau+transgenic+mice%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Detrimental effects of diet-induced obesity on tau pathology are independent of insulin resistance in tau transgenic mice.</a> Diabetes. 62, 1681-1688 (2013).</li>
<li>Deramecourt, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical%2C+neuropathological%2C+and+biochemical+characterization+of+the+novel+tau+mutation+P332S%5BTitle%5D)+AND+%22J+Alzheimers+Dis%22%5BJournal%5D)">Clinical, neuropathological, and biochemical characterization of the novel tau mutation P332S.</a> J Alzheimers Dis. 31, 741-749 (2012).</li>
<li>Birdsill, A. C., Walker, D. G., Lue, L., Sue, L. I., Beach, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postmortem+interval+effect+on+RNA+and+gene+expression+in+human+brain+tissue%5BTitle%5D)+AND+%22Cell+Tissue+Bank%22%5BJournal%5D)">Postmortem interval effect on RNA and gene expression in human brain tissue.</a> Cell Tissue Bank. 12, 311-318 (2011).</li>
<li>Bell, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+of+a+twenty-first+century+brain+bank%3A+experience+in+the+BrainNet+Europe+consortium%5BTitle%5D)+AND+%22Acta+Neuropathol%22%5BJournal%5D)">Management of a twenty-first century brain bank: experience in the BrainNet Europe consortium.</a> Acta Neuropathol. 115, 497-507 (2008).</li>
<li>Crecelius, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+quantitative+post-mortem+changes+in+the+gray+matter+of+the+human+frontal+cortex+proteome+by+2-D+DIGE%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Assessing quantitative post-mortem changes in the gray matter of the human frontal cortex proteome by 2-D DIGE.</a> Proteomics. 8, 1276-1291 (2008).</li>
<li>Swatton, J. E., Prabakaran, S., Karp, N. A., Lilley, K. S., Bahn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+profiling+of+human+postmortem+brain+using+2-dimensional+fluorescence+difference+gel+electrophoresis+%282-D+DIGE%29%5BTitle%5D)+AND+%22Mol+Psychiatry%22%5BJournal%5D)">Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE).</a> Mol Psychiatry. 9, 128-143 (2004).</li>
<li>Viswanathan, S., Unlu, M., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-dimensional+difference+gel+electrophoresis%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Two-dimensional difference gel electrophoresis.</a> Nat Protoc. 1, 1351-1358 (2006).</li>
<li>Tannu, N. S., Hemby, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-dimensional+fluorescence+difference+gel+electrophoresis+for+comparative+proteomics+profiling%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling.</a> Nat Protoc. 1, 1732-1742 (2006).</li>
</ol>

Authors have no conflict of interest to declare.

Discovering pathological expression changes of proteins, search for biomarkers and the modulation of potential pathways for pharmacological targets are among the aims of the neuroproteomics approaches30. Amid the emerging tools, the 2DE field adds a promising expectative. Nevertheless, a consensus should be reached in order to minimize variability and increase reproducibility in the experiments. In line of this idea a standardized protocol to perform 2D-DIGE (Figure 2) and the subsequent 2DE i...

Mental and neurological disorders represent 13% of the global burden of disease, new challenges like pathophysiological mechanisms, risk factors and prodromal biomarkers must be explored1. In line with this objective, proteomics studies of the human brain become indispensable to uncover molecular pathways involved in processes like memory, behavior, emotions and neuronal plasticity for instance, not only for physiological but also for pathological conditions. Therefore, the use of animal models and more specifically the transgenic mice, brings a wide range of possibilities to mimic the etiology of human neurodegenerative disorders2.
Proteomics approaches are nowadays available in order to accomplish these new perspectives in the neuroscience field. Two-dimensional gel electrophoresis (2DE) is a potent and likely simple method that enables to compare the proteome of a wide range of samples. Moreover, it is also a powerful method to isolate a protein from a complex mixture in order to identify and further analyze by mass spectrometry. This technique essentially consists in two successive steps: 1) protein separation according to their isoelectric point (pI) by isoelectrofocusing (IEF). More precisely, an electric potential is applied across the immobiline acrylamide strips among a pH gradient and then proteins will migrate and focus on a determined pI in function of their global net charge. 2) Isoelectrically focused proteins are denatured and negatively charged by the addition of sodium dodecyl sulfate (SDS), thereby proteins in their first structure are separated depending on their apparent molecular weight (MW) by SDS-PAGE3. These two distinct properties allow us to tackle a double value to go further in the study of the proteome. On one hand this approach offers the possibility to perform a quantitative analysis using minimal dyes 2D-DIGE method, and on the other hand a qualitative analysis by mini-2DE coupled to western blotting.
Quantitative analysis by 2D-DIGE bestows protein expression changes all over the sample proteome. Briefly, samples are labeled with three cyanines (Cy2, Cy3 and Cy5) emitting at three distinct wavelengths (blue, green and red). These fluor minimal dyes containing N-hydroxy succinimidyl ester group react with the &#949;-amino group of lysines residues of proteins resulting in covalent amide bonds4. Lysine residues are labeled only between 1-3% and thus preventing multiple labels addition per protein and major net charge modifications5,6. Cy3 and Cy5 are often used to label two independent samples while Cy2 tags a mix of equal proportion of the samples to compare. The two main advantages are that all labeled samples are mixed and IEF and SDS-PAGE are carried out in one gel at once for each step, avoiding the inter variability among experiments due to gels comparison. Moreover, it presents a high detection threshold of around 1 femtomole of protein7. Gels are scanned and 2D software compares the 2D gel fluorescence images, where Cy2 serves as an internal standard allowing for identification of statistical differences among the spots for their posterior identification by mass spectrometry. The 2D analysis software using the internal standard achieves a fast detection of less than 10% of differences between samples with more than 95% of statistical confidence8.
Qualitative analysis by mini-2DE is a crucial step for protein characterization. The principle is the same as previously described for pI and MW separation, but in this case proteins are transferred from a small polyacrylamide gel to a membrane and immunoblotting is performed afterwards. While one dimension gel electrophoresis provides changes in protein expression for one or several protein epitopes in function of the antibody, the information of mini-2DE endows with two additional parameters. Firstly, the protein isovariants change in function of the pI, indicating that post-translational modifications might take place. Secondly, mass spectrometry identification may be indicative of plausible zymogens and catabolic products of proteins. Therefore, modifications observed by 2D-DIGE are likely indicative of the mechanisms underlying changes in global proteome profile. Alignments of immunoblots among several samples for the same protein epitope/s by mini-2DE may reflect acidity changes shedding light into post-translational variations slightly observed or even not by monodimensional immunoblotting9,10. Moreover, this analysis informs about the potential cleavage sites due to the knowledge of the pI and MW of the metabolic residues11,12.
The combination of these two techniques provides a complementary proteomics analysis. On one hand 2D-DIGE affords a type of differences allowing a precise isolation of polypeptides whose expression is different. These differences consist essentially into the appearance or disappearance of a spot or increase/decrease of intensity of a given one by software analysis of the fluorescent gels. However, these observations by themselves are unlikely to explain the nature of the modification observed. For these reasons, once the polypeptide is isolated and identified by mass spectrometry, the use of mini-2DE enables to precisely confirm 1) the identity of the protein isolated and 2) the nature of the difference: change in the isovariant/isoform level of expression, post-translational modifications and cleavage processes for instance. However, it is necessary to develop a starting lysis buffer both compatible with 2D-DIGE and with mini-2DE in order to limit the potential dispersions resulting from the use of extraction protocols that are very dissimilar.
In the present article, we described an adapted protocol for the preparation, extraction and performance of 2D-DIGE and mini-2DE techniques for brain proteins coming from human and mouse tissue.

<table><tbody><tr><td>CyDye DIGE FLUOR MIN KIT 5 nmol 1 * 5 Nmo</td><td>GE Healtcare</td><td>25-8010-65</td><td></td></tr><tr><td>Immobiline DryStrip</td><td>GE Healtcare</td><td></td><td>Reference in function of the pH interval/size</td></tr><tr><td>IPG Buffer, pH X-X</td><td>GE Healtcare</td><td></td><td>Reference in function of the pH interval desired</td></tr><tr><td>AMBERLITE IRN-150L MIXED BED RESIN 1&amp;nbsp;</td><td>GE Healtcare</td><td>551797N</td><td></td></tr><tr><td>DRYSTRIP COVER FLUID IMMOBILINE 1 * 1 l</td><td>GE Healtcare</td><td>17-1335-01</td><td></td></tr><tr><td>KIT BOX IPG 1 * 1 KIT</td><td>GE Healtcare</td><td>28-9334-92</td><td>Plastic box to make the passive rehydration&amp;nbsp;</td></tr><tr><td>MILLEX GS Filter</td><td>Millipore</td><td>SLGS033SB</td><td></td></tr><tr><td>Criterion XT Precast Gels 13.3 x 8.7 cm (W x L)</td><td>Bio-Rad</td><td></td><td>Reference in function of the MW to separate</td></tr><tr><td>IPGphor III Isoelectric Focusing Unit</td><td>GE Healtcare</td><td>11-0033-64</td><td></td></tr><tr><td>Ettan DALTsix Large Electrophoresis System</td><td>GE Healtcare</td><td>80-6485-08</td><td></td></tr><tr><td>OneTouch 2D Gel SpotPicker 1.5 mm&amp;nbsp;</td><td>Gel Company</td><td>P2D1.5</td><td></td></tr></tbody></table>

consensus brain-derived protein, extraction protocol for the study of human and murine brain proteome using both 2d-dige and mini 2de immunoblotting

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

Proteomics on brain tissue remains challenging since no ideal buffer exists to recover 100% of proteins, especially membrane-associated or cytoskeleton proteins. The first set of experiments were focused on the search for a suitable lysis buffer compatible with the two approaches and which enables to recover a large panel of protein. Thus, three lysis buffers were assayed to determine the most appropriate one. Firstly, it was used the common biochemical and molecular biology buffer Tris-HCl20 at 10 mM with 1% ...

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

The overall goal of the following experiment is to extract protein from human and mouse brain tissue for proteomics analysis. This is achieved by using a common lysis buffer to obtain the same protein extraction adapted for two dimensional electrophoresis approaches. Next two dimensional fluorescence difference gel electrophoresis is used to separate proteins for mass spectrometry identification.This is followed by a miniature 2D gel electrophoresis with immuno blotting to identify post-translational modifications. The results can show global protein expression. Host translational changes all ligamentization and catalytic products based on the differences in isoelectric point and molecular weight.The combination of 2D dash and 2D monolo can help us to answer key question in the neuro proteomics field by simultaneously providing information on expression changes. Postal modification and catabolism in products Begin with harvesting and homogenizing the brain tissue. Prepare the tissue at 10%weight to volume in extraction buffer for human brain tissue.Homogenize the tissue using a glass Pipetter, however, use a Teflon homogenizer for rodent tissue to disintegrate either homogenate. Sonicate it at 60 hertz using 30 half second pulses. Next, determine the protein concentration with a Bradford assay using BSA as a standard.Now gather the solutions for chloroform methanol precipitation in an ice bucket. Plan to collect about 125 micrograms for 11 centimeter IPG strips or about 1.25 milligrams for 18 centimeter IPG strips. Once the solutions are at four degrees Celsius, perform the precipitation completely on ice.First, add three volumes of methanol, then one of chloroform. Shake this mixture. Next, add three volumes of cold water.Vortex, this mixture for one minute centrifuge the sample tube at 12, 000 Gs for 30 minutes at four degrees Celsius. Aspirate and discard the supernatant. Then add three volumes of methanol vortex and spin down the proteins in the same manner.Dry the collected protein pellet under nitrogen gas before 2D DIGE. Re suspend the protein in 2D buffer at 2.5 milligrams per milliliter. Then sonicate the samples as done before until they are used.Store them at minus 80 degrees Celsius before beginning this step and after protein precipitation. It is necessary to determine the protein concentration once more by Bradford Assay. Following the same procedure as shown earlier before psy dye labeling.The quality of the protein sample is verified by SDS page for that purpose. Dilute 15 micrograms of protein in LDS and he it to 37 degrees Celsius in a heat bath or block. Then run them on a poly acrylamide gel stain the gel with kumasi blue solution for at least an hour.Then detain the gel overnight in 7%acetic acid, 10%ethanol solution. Once the protein sample quality is established, the next day the SD labeling procedure is started. First measure the pH of the lysate, which should be basic to neutral.The optimal pH is 8.6 for subsequent reactions. Next, set up the dye conjugation reactions for each protein sample. Mix 50 micrograms with 400 picomoles of S3 dye and 50 micrograms with 400 picomoles of S five dye.Do this in quadruplex for a total of eight D coupling reactions per sample. For each tested sample, make a complimentary set of DI coupling reactions using protein from the control such as from a diseased brain. Then for every pair of reactions, set up an internal control reaction with 400 picomoles of CY two and an even representation of all proteins totaling 250 micrograms.SAI labeling is achieved by incubating all the reaction mixtures at four degrees Celsius in the dark for an hour. After the incubation, combine all the sets of three different cyan reactions into 150 microliter volumes to each set of reactions at 200 microliters of 2D buffer. Then proceed with the 2D electrophoresis steps.Begin with preparing unlabeled protein solutions for the preparative gel from which protein spots will be excised for each sample and for the positive control. Aliquot 300 to 350 micrograms of unlabeled protein into 2D buffer and allow them to rehydrate in contact with the IPG strip. Next load the IPG strips transfer the labeled and the unlabeled protein to a rehydration.Well then cover each well with an IPG reaction strip. Follow this with an overlay of mineral oil and allow the strips to passive passively rehydrate with the protein solution overnight at no more than 25 degrees Celsius. The next day, carry out isoelectric focusing of the IPG strips.Then remove the oil and store the IPG strips at minus 20 degrees Celsius to equilibrate the IPG strips. Submerge them in equilibration buffer for 15 minutes. Then transfer the strips to 4.7%IDO acetamide in equilibration buffer, lacking DTT while bathing the strips.Use a large 2D gel system to pour four 12%SDS page gels per sample in low fluorescence glass plates for the labeled proteins. Also pour one 12%SDS page gel in 1.5 millimeter thick standard glass plates for each of the unlabeled proteins. These are the preparative gels.When the gels are cooled, transfer the strips to the top of the gels and overlay the strips with 0.5%Aros once cooled, run the gels at 2.5 watts at four degrees Celsius overnight and analyze them the next day. Many 2D reactions can be carried out with protein samples used for the preparative gels. Measure out less than 100 micrograms of sample into 200 microliters of 2D buffer.Then vortex the sample mixtures vigorously and give them a quick spin. Load the samples onto 11 centimeter IPG strips and allow them to passively rehydrate overnight covered in mineral oil the following day. Perform the isoelectric focusing.Then move the strips through three baths of equilibration buffer for 15 minutes per bath. The strips are then run on 2D SDS page gels. Layer each strip onto a precast gel of the appropriate molecular weight.Poly acrylamide for the protein of interest. Later, transfer the gels to NITROCELLULOSE PVDF membrane and analyze them with antibodies in development of this protocol. Three different lysis.Buffers were tested. Firstly, a common biochemical and molecular biology buffer. Tris SDS was tested.Tris SDS had poor resolution compared to another tested buffer UTS. The third buffer to de was ruled out as testable samples could not be prepared with it. Next, human and mouse brain proteomes were investigated.Human control cortex samples and ad cortex samples were compared. Mouse brain proteins from mice with AD like tau pathology were also viewed from the human sample. SCI two was looked at independently as was S SI three and sci five for each D.The high spot resolution made alignment easy.Qualitative mini 2D analysis was used to investigate post-translational modifications and a ligamentization alpha synuclein in ad tissue was found to have a normal profile, but also found as a dimer marked by an asterisk. Arrows marked where alpha synuclein was found. Ubiquitinated amyloid precursor protein was also viewed by mini 2D.Sporadic ad was compared with familial ad. In familial ad beta amyloid one to 42 and ISO variance are lower in concentration because liga forms were found at eight kilodaltons. After watching this video, you should have a good understanding of how to study the proteome in order to compare complex samples by measuring protein expression modifications.

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Neuroscience

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An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

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Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

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Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Learn a consensus brain protein extraction protocol for proteomics using human and murine tissue with 2D-DIGE and mini 2DE immunoblotting. This method enables reliable identification of post-translational modifications.

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting (Video) | JoVE Journal

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

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