Name: identification and characterization of protein glycosylation using specific endo- and exoglycosidases
Uploaded: 2011-12-26T13:00:00
Duration: 9 min 54 s
Description: Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

1. Enzymatic deglycosylation
<ol>
 <li>Use PCR tubes to minimize water loss due to evaporation. Label one set of tubes 1 to 7.</li>
 <li>Thaw the 10X G7 buffer, the 10X glycoprotein denaturing buffer, and the 10% NP-40 and gently tap the tubes to mix the contents. Keep at room temperature.</li>
 <li>Place the enzyme-containing vials on ice. Try to minimize thaw/freeze cycles.</li>
 <li>Dissolve the contents of the hCG&beta; vial (150 &mu;g) in 600 &mu;l of dH2O and keep on ice.</li>
 <li>Prepare 1 ml of 1X G7 buffer by diluting the 10X stock in dH2O.</li>
 <li>Dilute 0.5 &mu;l of PNGase F in 25 &mu;l 1X G7 buffer and keep on ice.</li>
 <li>Prepare the exoglycosidase mix (EG mix) by combining 2 &mu;l each of &alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &beta;1-3 Galactosidase, and &alpha;1-3, 6 Galactosidase.</li>
 <li>Set up PCR tubes as indicated:</li>
</ol>




<table border="1">
 <tbody>
 <tr>
 <td>Tube/sample#</td>
 <td>1</td>
 <td>2</td>
 <td>3</td>
 <td>4</td>
 <td>5</td>
 <td>6</td>
 <td>7</td>
 </tr>
 <tr>
 <td>hCG&beta; (0.25mg/ml)</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 <td>--</td>
 <td>--</td>
 <td>--</td>
 </tr>
 <tr>
 <td>10X Glycoprotein&nbsp;Denaturing&nbsp;Buffer</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>--</td>
 <td>--</td>
 <td>--</td>
 <td>--</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 </tr>
 </tbody>
</table>
<ol start="9">
 <li>Cap the tubes, mix gently and place in the thermocycler, close the lid and denature the proteins by incubating 10 minutes at 94&deg;C, followed by a 4&deg;C hold (using PCR tubes in a thermocycler greatly prevents evaporation in small volume samples).</li>
 <li>Remove the tubes from thermocycler and centrifuge to remove any visible condensation.</li>
 <li>Add the following reagents as indicated:</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td>Sample#</td>
 <td>1</td>
 <td>2</td>
 <td>3</td>
 <td>4</td>
 <td>5</td>
 <td>6</td>
 <td>7</td>
 </tr>
 <tr>
 <td>10% NP-40</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 </tr>
 <tr>
 <td>10X G7 Buffer</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 </tr>
 <tr>
 <td>PNGase F 1:50 dil.</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>--</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>--</td>
 <td>--</td>
 </tr>
 <tr>
 <td>Deglycosylation Mix</td>
 <td>--</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>2&mu;l</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>2&mu;l</td>
 </tr>
 <tr>
 <td>EG mix</td>
 <td>--</td>
 <td>--</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>--</td>
 <td>--</td>
 <td>2&mu;l</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>10&mu;l</td>
 <td>8&mu;l</td>
 <td>8&mu;l</td>
 <td>6&mu;l</td>
 <td>8&mu;l</td>
 <td>8&mu;l</td>
 <td>6&mu;l</td>
 </tr>
 <tr>
 <td>Total reaction vol.</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 </tr>
 </tbody>
</table>
<ol start="12">
 <li>Close the PCR tubes using new caps (discard the used ones since they do not fit properly after one incubation cycle).</li>
 <li>Mix the tubes by gently tapping 4 times and then spin the contents down.</li>
 <li>Place the tubes in the thermocycler and incubate at 37 &deg;C for 4 hours then cool the samples to 4 &deg;C.</li>
</ol>
2. SDS-PAGE of deglycosylated samples
<ol>
 <li>Prepare 130 &mu;l fresh 3X reducing SDS loading buffer by adding 4 &mu;l of 1.25M DTT.</li>
 <li>Add 12.5 &mu;l of the prepared 3X reducing SDS loading buffer to each sample.</li>
 <li>Close the tubes with new caps and gently tap the tubes to mix.</li>
 <li>Incubate the tubes in a thermocycler at 94&deg;C for 5 minutes and then cool to 4 &deg;C.</li>
 <li>Load 30 &mu;l of each sample and 10 &mu;l of the protein marker on a 10-20% Tris-Glycine gel. Save the remainder of the sample for part 3.1. Load 10&mu;l of ColorPlus Prestained Protein Marker.</li>
 <li>Electrophorese the gel at 130 volts at room temperature until the dye front is near the bottom of the gel.</li>
 <li>When the gel has finished running, remove the gel from the cast and place it in a small plastic box with enough Coomasie blue stain to cover the gel.</li>
 <li>Stain the gel for 1 hour with gentle agitation.</li>
 <li>Wash the gel three times for 30 minutes in 50 ml of destain solution.</li>
 <li>Record the images using a white light transilluminator or scanner. Alternatively, the gel can be dried between sheets of cellophane in a frame.</li>
</ol>
3. Pro-Q Emerald 300 for detection of glycosylated proteins in SDS-PAGE gels
<ol>
 <li>In parallel with the gel in 2.1), load the remainder of the samples on a 10-20% Tris-Glycine gel. Load 10&mu;l of ColorPlus Prestained Protein Marker.</li>
 <li>While the gel is running dissolve the Pro-Q Emerald reagent with DMF and prepare the stock Fix, Wash and Oxidizing solutions for the Pro-Q Emerald 300 stain following the product manual provided with the kit.</li>
 <li>When the electrophoresis is complete, remove the gel from cast and place it in a plastic box.</li>
 <li>Fix the gel by adding 100 ml of the Fix solution and leaving it overnight at room temperature with gentle agitation.</li>
 <li>Wash the gel with 100 ml of the Wash solution for 10 to 20 minutes at room temperature with gentile agitation. Repeat the wash with fresh Wash solution.</li>
 <li>Oxidize the carbohydrates by incubating the gel with gentle agitation for 30 minutes in 25 ml of Oxidizing solution.</li>
 <li>Wash the gel as described in step 3.5.</li>
 <li>While the gel is washing prepare fresh Pro-Q Emerald 300 stain by adding 500 &mu;l of the Pro-Q Emerald 300 reagent solution dissolved in step 3.2 to 25 ml of staining buffer provided in the kit.</li>
 <li>Stain the gel by adding 25 ml of the stain prepared in step 3.8 and incubating in the dark with gentle agitation for 90 to 120 minutes.</li>
 <li>Repeat the two wash steps described in step 3.5.</li>
 <li>Record the images with an UV transilluminator at 300nm. Use the 80 kDa marker, which is labeled with Pro-Q Emerald Green, to overlap the UV image to a white light picture of the gel showing the prestained ladder.</li>
 <li>Compare the images of the Coomasie stained gel in step 2.10 with the Pro-Q Emerald stained gel from step 3.11.</li>
</ol>
4. Representative results
The changes in protein migration after enzymatic deglycosylation are shown in Figure 2. Compare the control sample (panel A, lane 1) with the PNGase F treatment (removal of N-glycans, lane 2), and Deglycosylation Mix (PNGase F; plus endo-and exoglycosidases to remove O-glycans, lane 3). No further reduction in size is seen after digesting with additional glycosidases (lane 4). Besides a change in mass, bands become sharper as glycans are removed. A band running under the 17 kDa marker (asterisk) represents the fully deglycosylated hCG&beta; polypeptide (MW: 16 kDa). Other bands might derive from incomplete deglycosylation or from the multiple unidentified proteins present in the hCG&beta; sample (see lane 1). Lanes 5 to 7 (controls) show the bands corresponding to the glycosidases.
The glycoprotein staining by Emerald Green is shown in panel B. This reagent oxidizes and stains all glycans present in a protein molecule. Therefore the intensity of the signal decreases as hCG&beta; is enzymatically deglycosylated (lane 1 to lane 4). The residual signal in lanes 3 and 4 indicate the presence of glycan motifs, which are resistant to the enzymes used. The additional glycosidases used in lane 4 remove a few extra sugar residues: the protein migration is the same, but a slight reduction in intensity in staining can be seen. Resistant sugar moieties were not present in all protein species: some bands were not detected by Emerald Green (absent in the UV image, in brackets), indicating they were extensively deglycosylated. Additional data support the conclusion that hCG&beta; is heterogeneously glycosylated. The lower band on lane 2 (arrow) is faint on the Emerald Green image, while the upper band on lane 2 is bright, indicating that many glycans groups are still present. These data support the conclusion that recombinant hCG&beta; expressed in mouse cells contains multiple glycoforms9 . These different glycoforms are due to the inherent heterogeneity of glycosylation where some polypeptides do not receive a glycan in each consensus site and/or some glycans are extended while others even on the same protein are not.
<img alt="Figure 1" src="/files/ftp_upload/3749/3749fig1.jpg" /> 
Figure 1. Typical glycosylation patterns for secreted or cell-surface glycoproteins.
<img alt="Figure 2" src="/files/ftp_upload/3749/3749fig2.jpg" /> 
Figure 2. SDS-PAGE gels showing enzymatic deglycosylation of hCG&beta;. Panel A shows a Coomasie blue staining while Panel B shows the results of Pro-Q Emerald 300 for visualization of glycosylated proteins. Sample number: 1, hCG&beta; control; 2, PNGase F digestion; 3, Deglycosylation Mix digestion; 4, Deglycosylation Mix plus exoglycosidases digestion, samples 5 to 7 are the reagent controls (O-Glyc, O-Glycosidase; GNA, &beta;-N-Acetylglucosaminidase; Gal/Fuc, &beta;1-3 Galactosidase, &alpha;1-3,6 Galactosidase, and &alpha;1-2 Fucosidase; GalNA, &alpha;-N-Acetylglalactosaminidase; Neu, Neuraminidase; PNGase; PNGase F)

<ol>
	<li>Spiro, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+glycosylation:+nature,+distribution,+enzymatic+formation,+and+disease+implications+of+glycopeptide+bonds.">Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.</a> Glycobiology. 12 (4), 43-56 (2002).</li><li>Ohtsubo, K., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+Cellular+Mechanisms+of+Health+and+Disease.">Glycosylation in Cellular Mechanisms of Health and Disease.</a> Cell. 126, 855-867 (2006).</li><li>Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M., Dwek, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+glycosylation+on+the+biological+function+and+structure+of+human+immunoglobulins.">The impact of glycosylation on the biological function and structure of human immunoglobulins.</a> Annual Review of Immunology. 25, 21-50 (2007).</li><li>Mitra, N., Sinha, S., Ramya, T. N. C., Surolia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-linked+oligosaccharides+as+outfitters+for+glycoprotein+folding,+form+and+function.">N-linked oligosaccharides as outfitters for glycoprotein folding, form and function.</a> Trends in Biochemical Sciences. 31, 156-163 (2006).</li><li>Carlsen, R. B., Bahl, O. P., Swaminathan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chorionic+gonadotropin.+Linear+amino+acid+sequence+of+the+beta+subunit.">Human chorionic gonadotropin. Linear amino acid sequence of the beta subunit.</a> Journal of Biological Chemistry. 248 (19), 6810-6812 (1973).</li><li>Tarentino, A. L., Plummer, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deglycosylation+of+Asparagine-linked+Glycans+by+PNGaseF.">Deglycosylation of Asparagine-linked Glycans by PNGaseF.</a> Trends in Glycoscience and Glycotechnology. 5 (23), 163-170 (1993).</li><li>Tretter, V., Altmann, F., Marz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine+amidase+F+cannot+release+glycans+with+fucose+attached+&#945;1-3+to+the+asparagine-linked+N-acetylglucosamine+residue.">Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached &#945;1-3 to the asparagine-linked N-acetylglucosamine residue.</a> European Journal of Biochemistry. 199, 647-652 (1991).</li><li>Koutsioulis, D., Landry, D., Guthrie, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+endo-&#945;-N-acetylgalactosaminidases+with+broader+substrate+specificity.">Novel endo-&#945;-N-acetylgalactosaminidases with broader substrate specificity.</a> Glycobiology. 18 (10), 799-805 (2008).</li><li>Thakur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+glycoforms+of+the+intact+alpha+subunit+of+recombinant+human+chorionic+gonadotropin+by+high-resolution+capillary+electrophoresis-mass+spectrometry.">Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.</a> Analytical Chemistry. 81 (2), 8900-8907 (2009).</li><li>Park, S., Lee, M. R., Shin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+microarrays+as+powerful+tools+in+studies+of+carbohydrate-mediated+biological+processes.">Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes.</a> Chemical Communications. 37, 4389-4399 (2008).</li><li>Zachara, N. E., Vosseller, K., Hart, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+analysis+of+proteins+modified+by+O-linked+N-acetylglucosamine.">Detection and analysis of proteins modified by O-linked N-acetylglucosamine.</a> Current Protocols in Molecular Biology. 95, 17-17 (2011).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2008).</li></ol>

The authors are employed by New England Biolabs, which produces many of the reagents used in this article.

The method described here using enzymatic deglycosylation and SDS-PAGE can provide valuable information about the glycosylation state of a protein of interest, while glycan-specific reagents facilitate the interpretation of the data. This protocol is intended for the initial studies of protein glycosylation and it is particularly suited for secretory and membrane glycoproteins from mammalian cells: the enzymes chosen in this case will specifically remove all N-glycans, and or N-glycans plus and the most common sugars extending and forming the core of O-glycans. Glycosidases have the additional advantage of being mild, compared with chemical deglycosylation methods, preserving the integrity of both sugars and protein backbone.
To elucidate the rate of occupancy (which amino acids are glycosylated), extent of glycosylation, or to determine the fine structure of glycans, more sophisticated techniques such as mass spectrometry, liquid chromatography or NMR are required.
Because of its simplicity, several steps in this protocol can be adjusted, substituted, and/or combined to accommodate various experimental needs. However, in order to obtain results that can be clearly interpreted it is important to understand its strengths and limitations. First, the specificity and purity of the glycosidases are crucial: only well-characterized enzymes tested to be free of proteases and other contaminating activities should be used. Unfortunately, there is no standard enzyme unit definition for glycosidases; the user should determine the appropriate substitution according to the manufacturer's specifications. Second, a careful choice of detection is necessary: a) Protein staining reagents are useful only if deglycosylation results in a significant shift in molecular mass. Such a clear result as shown here is not always obtained. In other cases, we have seen abnormal migration after deglycosylation (far from the predicted molecular weight, or even slower migration). This phenomenon is not well understood, but it can be said that any change in migration is evidence that the protein has been deglycosylated. b) Sugar detection with antibodies presents unique challenges limiting their applicability. It has been very difficult to generate general anti-glycan antibodies; they are usually raised against a complex glycan target, which restricts their use. Moreover, several monoclonal anti-glycan antibodies display undesired cross reactivity10. c) Lectins (proteins with intrinsic sugar affinity) are well suited for sugar detection, plus they give insight on glycan structure. However, not all of them have a narrow specificity; and many are only partially characterized (meaning that they could have unknown affinities). As a consequence positive lectin staining provides indication, not proof, of the presence of a given sugar. d) Chemical labeling kits (based on periodate oxidation of sugars) are the method of choice to stain all glycoproteins, and thus well suited to follow deglycosylation.
When processing an unknown sample, it is a good practice to include glycoprotein controls. Fetuin is a readily available N- and O-glycoprotein; chorionic gonadotropin (both subunits) is also a good choice. Bovine serum albumin (BSA) can be used as a negative control. However, it should be noted that some non-glycosylated proteins react slightly with the Pro-Q Emerald 300, particularly when used at high concentrations. Non-glycosylated molecular weight standards, such as the prestained protein marker used in this protocol, have the advantage of displaying sharp bands. Only by chance is one of these proteins (80 kDa) reactive to Pro-Q Emerald 300. Therefore, the user might want to run a glycoprotein standard ladder instead, such as Candy-Cane from Invitrogen.
Finally, simple in-gel detection of nucleocytosolic glycoproteins (which are modified with a single O-GlcNAc) is possible with the use of a monoclonal antibody, along &beta;-N-Acetylglucosaminidase for specificity control 11.The description of this technique is beyond the scope of this article, but it should be mentioned as glycosidases are useful tools for the study of many other glycoproteins and glycoconjugates present in cells. A comprehensive treatment of all known forms of glycosylation can be found in the second edition of the "Essentials of Glycobiology"; available free online at the NCBI Bookshelf (Bookshelf ID: NBK1908; PMID: 20301239)12.

<table border="1">
<tbody>
 <tr>
 <td>Name of the 			reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Human chorionic gonadotropin &beta;</td>
 <td>Sigma Aldrich</td>
 <td>C6572</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>PCR Tubes</td>
 <td>VWR</td>
 <td>20170-004</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>PCR Thermocycler</td>
 <td>Applied Biosystems</td>
 <td>4359659</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>PNGase F</td>
 <td>New England Biolabs</td>
 <td>P0704</td>
 <td>Supplied with buffers </td>
 </tr>
 <tr>
 <td>Protein Deglycosylation Mix</td>
 <td>New England Biolabs</td>
 <td>P6039</td>
 <td>Supplied with buffers</td>
 </tr>
 <tr>
 <td>&alpha;-N-Acetylglalactosaminidase</td>
 <td>New England Biolabs</td>
 <td>P0734</td>
 <td>Supplied with buffer</td>
 </tr>
 <tr>
 <td>&alpha;1-2 Fucosidase</td>
 <td>New England Biolabs</td>
 <td>P0724</td>
 <td>Supplied with buffer</td>
 </tr>
 <tr>
 <td>&alpha;1-3,6 Galactosidase</td>
 <td>New England Biolabs</td>
 <td>P0731</td>
 <td>Supplied with buffer</td>
 </tr>
 <tr>
 <td>&beta;1-3 Galactosidase</td>
 <td>New England Biolabs</td>
 <td>P0726</td>
 <td>Supplied with buffer</td>
 </tr>
 <tr>
 <td>10X Buffer G7 (500 mM Sodium Phosphate)</td>
 <td>New England Biolabs</td>
 <td>---</td>
 <td>Supplied with PNGaseF or Degl Mix</td>
 </tr>
 <tr>
 <td>10X 			Glycoprotein Denaturing Buffer
 (5% SDS, 400 mM DTT)</td>
 <td>New England Biolabs</td>
 <td>---</td>
 <td>Supplied with PNGaseF or Degl Mix</td>
 </tr>
 <tr>
 <td>10% NP-40</td>
 <td>New England Biolabs</td>
 <td>---</td>
 <td>Supplied with PNGaseF or Degl Mix</td>
 </tr>
 <tr>
 <td>3X SDS loading buffer (187.5 mM Tris-HCl 			pH 6.8, 6% SDS, 30% glycerol, 0.03% bromophenol blue)</td>
 <td>New England Biolabs</td>
 <td>B7703</td>
 <td>Supplied with 1.25M DTT</td>
 </tr>
 <tr>
 <td>ColorPlus Prestained Protein Marker</td>
 <td>New England Biolabs</td>
 <td>P7709</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>10-20% Tris-Glycine Multigel</td>
 <td>Cosmo Bio Co.</td>
 <td>DCB-414893</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Cassette Electrophoresis Unit</td>
 <td>Cosmo Bio Co. </td>
 <td>DCB-303111</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Electrophoresis 			Power Supply 
 EPS 301</td>
 <td>GE Healthcare</td>
 <td>18-1130-01</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Pro-Q Emerald 300 Glycoprotein Stain Kit</td>
 <td>Invitrogen</td>
 <td>P-21857</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Brilliant Blue R</td>
 <td>Sigma Aldrich</td>
 <td>B0149</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>AlphaImager HP System</td>
 <td>Cell Biosciences</td>
 <td>92-13823-00</td>
 <td>&nbsp; </td>
 </tr>
 </tbody>
</table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

Research

JoVE Journal

Biology

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.