The antibodies against MHC I (A4.840) and MHCIIa (A4.74) used in this study were developed by Dr. H. M. Blau and the antibody against MHCIIx (6H1) was developed by Dr. C. A. Lucas and obtained from the Developmental Studies Hybridoma Bank (DSHB), thanks to the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, IA). We thank Victoria L. Wyckelsma for providing the human muscle samples for this study. The majority of the images in Figure 1 was sourced from BioRender.com.
Funding: 
This study received no external funding.

MyDoBID- A Novel Technique for Identifying Muscle Fiber Types Efficiently

<ol>
	<li>Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+types+in+mammalian+skeletal+muscles.">Fiber types in mammalian skeletal muscles.</a> Physiological Reviews. 91 (4), 1447-1531 (2011).</li><li>Bloemberg, D., Quadrilatero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+myosin+heavy+chain+expression+in+rat,+mouse,+and+human+skeletal+muscle+using+multicolor+immunofluorescence+analysis.">Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis.</a> PLoS One. 7 (4), e35273 (2012).</li><li>Wyckelsma, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+specific+differences+in+the+protein+abundances+of+GAPDH+and+Na(+),K(+)-ATPase+in+skeletal+muscle+from+aged+individuals.">Cell specific differences in the protein abundances of GAPDH and Na(+),K(+)-ATPase in skeletal muscle from aged individuals.</a> Experimental Gerontology. 75, 8-15 (2016).</li><li>Morales-Scholz, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+fiber+type-specific+autophagy+responses+following+an+overnight+fast+and+mixed+meal+ingestion+in+human+skeletal+muscle.">Muscle fiber type-specific autophagy responses following an overnight fast and mixed meal ingestion in human skeletal muscle.</a> American Journal of Physiology Endocrinology and Metabolism. 323 (3), e242-e253 (2022).</li><li>Tripp, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+and+fibre+type-dependent+nature+of+calcium-handling+protein+responses+to+sprint+interval+exercise+in+human+skeletal+muscle.">Time course and fibre type-dependent nature of calcium-handling protein responses to sprint interval exercise in human skeletal muscle.</a> The Journal of Physiology. 600 (12), 2897-2917 (2022).</li><li>Frankenberg, N. T., Mason, S. A., Wadley, G. D., Murphy, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+cell-specific+differences+in+type+2+diabetes.">Skeletal muscle cell-specific differences in type 2 diabetes.</a> Cellular and Molecular Life Sciences. 79 (5), 256 (2022).</li><li>Murphy, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+skeletal+muscle+calpain-3+by+eccentric+exercise+in+humans+does+not+result+in+its+translocation+to+the+nucleus+or+cytosol.">Activation of skeletal muscle calpain-3 by eccentric exercise in humans does not result in its translocation to the nucleus or cytosol.</a> Journal of Applied Physiology. 111 (5), 1448-1458 (2011).</li><li>Murphy, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+technique+to+measure+proteins+in+single+segments+of+human+skeletal+muscle+fibers:+fiber-type+dependence+of+AMPK-alpha1+and+-beta1.">Enhanced technique to measure proteins in single segments of human skeletal muscle fibers: fiber-type dependence of AMPK-alpha1 and -beta1.</a> Journal of Applied Physiology. 110 (3), 820-825 (2011).</li><li>Christiansen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast,+reliable+and+sample-sparing+method+to+identify+fibre+types+of+single+muscle+fibres.">A fast, reliable and sample-sparing method to identify fibre types of single muscle fibres.</a> Scientific Reports. 9 (1), 6473 (2019).</li><li>Skelly, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+skeletal+muscle+fiber+type-specific+responses+to+sprint+interval+and+moderate-intensity+continuous+exercise:+acute+and+training-induced+changes.">Human skeletal muscle fiber type-specific responses to sprint interval and moderate-intensity continuous exercise: acute and training-induced changes.</a> Journal of Applied Physiology. 130 (4), 1001-1014 (2021).</li><li>Bergstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+electrolytes+in+man.">Muscle electrolytes in man.</a> Scandinavian Journal of Clinical and Laboratory Investigation. (SUPPL. 68), 1-110 (1962).</li><li>Evans, W. J., Phinney, S. D., Young, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suction+applied+to+a+muscle+biopsy+maximizes+sample+size.">Suction applied to a muscle biopsy maximizes sample size.</a> Medicine &amp; Science in Sports &amp; Exercise. 14 (1), 101-102 (1982).</li><li>Wyckelsma, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+skeletal+muscle+mitochondrial+content+in+older+adults:+relationship+between+mitochondria,+fibre+type+and+high-intensity+exercise+training.">Preservation of skeletal muscle mitochondrial content in older adults: relationship between mitochondria, fibre type and high-intensity exercise training.</a> The Journal of Physiology. 595 (11), 3345-3359 (2017).</li><li>Bortolotto, S. K., Stephenson, D. G., Stephenson, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+type+populations+and+Ca2+-activation+properties+of+single+fibers+in+soleus+muscles+from+SHR+and+WKY+rats.">Fiber type populations and Ca2+-activation properties of single fibers in soleus muscles from SHR and WKY rats.</a> American Journal of Physiology Cell Physiology. 276 (3), C628-C637 (1999).</li><li>Murphy, R. M., Lamb, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+considerations+for+protein+analyses+using+antibody+based+techniques:+down-sizing+Western+blotting+up-sizes+outcomes.">Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes.</a> The Journal of Physiology. 591 (23), 5823-5831 (2013).</li><li>Lucas, C. A., Kang, L. H., Hoh, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monospecific+antibodies+against+the+three+mammalian+fast+limb+myosin+heavy+chains.">Monospecific antibodies against the three mammalian fast limb myosin heavy chains.</a> Biochemical and Biophysical Research Communications. 272 (1), 303-308 (2000).</li><li>MacInnis, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+mitochondrial+adaptations+in+human+skeletal+muscle+after+interval+compared+to+continuous+single-leg+cycling+matched+for+total+work.">Superior mitochondrial adaptations in human skeletal muscle after interval compared to continuous single-leg cycling matched for total work.</a> The Journal of Physiology. 595 (9), 2955-2968 (2017).</li></ol>

The authors have no conflicts of interest to declare.

Fiber collection 
Based on several years of experience, most researchers can master this technique; however, practice leads to faster and more efficient fiber collection for downstream analyses. To be able to isolate 30 single fiber segments of a quality for pooling, it is recommended that 50 fiber segments are collected per sample. Studying the fiber collection video carefully and after performing two practice sessions (~50 fibers per session) is recommended to achieve a reasonable standard. All fi...

Skeletal muscle is a heterogeneous tissue, with distinct cellular metabolic and contractile properties that are dependent on whether the cell (fiber) is slow twitch (type I) or fast twitch (type II). The fiber type can be identified by examining the myosin heavy chain (MHC) isoforms, which differ from each other in several ways, including contraction time, speed of shortening, and fatigue resistance1. Major MHC isoforms include type I, type IIa, type IIb, and type IIx and their metabolic profiles are either oxidative (type I and IIa) or glycolytic (IIx, IIb)1. The proportion of these fiber types varies in muscle type and between species. Type IIb is widely found in rodent muscle. Human muscles do not contain any type IIb fibers and consist predominantly of MHC isoforms type I and IIa fibers, with a small proportion of IIx fibers2. Protein expression profiles vary between different fiber types and can be altered with aging3, exercise4,5, and disease6.
Measuring cellular responses in different skeletal muscle fiber types is often overlooked or not possible due to the examination of muscle homogenates (a mix of all fiber types). Single-fiber western blot allows for the investigation of multiple proteins in individual muscle fibers7. This methodology has previously been utilized to produce novel and informative single-fiber characteristics that were not possible to obtain using homogenate preparations. However, there are some limitations of the original single-fiber western blot methodology, including the time-consuming nature, the inability to generate sample replicates, and the use of expensive, sensitive enhanced chemiluminescence (ECL) reagents. If fresh tissue is being used, this method is further limited due to the time constraints of needing to isolate individual fibers within a limited timeframe (i.e., 1-2 h). Fortunately, this restraint is mitigated by isolating single-fiber segments from freeze-dried tissue8. However, fiber collection from freeze-dried samples is limited by the size and quality of the biopsied tissue.
Fiber type identification using the dot blotting method9 has been significantly elaborated upon and expanded in this comprehensive protocol. Previously, it has been demonstrated that as little as ~2-10 mg of wet weight muscle tissue is adequate for freeze-drying and single-fiber MHC isoform protein analysis9. Christensen et al.9 used 30% of a ~1 mm fiber segment to detect the MHC isoform present by dot blotting, which was confirmed by western blotting. This work showed that by substituting western blotting with dot blotting, the overall costs were reduced by ~40-fold (for 50 fiber segments). Fibers were then &#34;pooled&#34; into type I and type II samples, which allowed for experimental replication9. Nevertheless, a limitation was that only two fiber type-specific samples were obtained: type I (MHCI positive) and type II (MHCII positive fibers), with type II samples containing a mixture of MHCIIa and MHCIIx6,10. Notably, the current protocol demonstrates how pure type IIx fibers can be identified and provides a highly detailed workflow (summarized in Figure 1), including troubleshooting strategies for common protocol issues.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x Denaturing buffer</td>
			<td>Make according to recipe</td>
			<td>Constituents can be sourced from Sigma-Aldrich or other chemical distributing companies</td>
			<td>&#160;1x denaturing buffer is made by diluting 3x denaturing buffer 1 in 3 v/v with 1X Tris-HCl (pH 6.8). Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>3x Denaturing buffer</td>
			<td>Make according to recipe</td>
			<td>Constituents can be sourced from Sigma-Aldrich or other chemical distributors</td>
			<td>3x denaturing buffer contains: 0.125M Tris-HCI, 10% glycerol, 4% SDS, 4 M urea, 10% 2-mercaptoethanol, and 0.001% bromophenol blue, pH 6.8.&#160; Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>N/A</td>
			<td>100% ethanol can be sourced from any company</td>
			<td>Diluted to 95% with ultra-pure H2O.</td>
		</tr>
		<tr>
			<td>Actin rabbit polyclonal antibody</td>
			<td>Sigma-Aldrich</td>
			<td>&#160; A2066</td>
			<td>Dilute 1 in 1,000 with BSA buffer.</td>
		</tr>
		<tr>
			<td>Analytical scales</td>
			<td>Mettler Toledo</td>
			<td>Model number: MSZ042101</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody enhancer</td>
			<td>Thermo Fischer Scientific</td>
			<td>32110</td>
			<td>Product name is Miser Antibody Extender Solution NC.</td>
		</tr>
		<tr>
			<td>Beaker (100 mL)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>5452</td>
			<td>Model name: Mini Spin.</td>
		</tr>
		<tr>
			<td>Blocking buffer: 5% Skim milk in Wash buffer.</td>
			<td>Diploma</td>
			<td>Store bought</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA buffer: 1 % BSA/PBST, 0.02 % NaN3</td>
			<td>BSA: Sigma-Aldrich&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; PBS: Bio-Rad Laboratories. NaN3 : Sigma-Aldrich&#160;</td>
			<td>BSA: A6003-25G&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 10x PBS: 1610780&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; NaN3: S2002</td>
			<td>Bovine serum albumin (BSA), Phosphate-buffered saline (PBS), and Sodium azide (NaN3). Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Cassette opening lever&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>4560000</td>
			<td>Used to open the precast gel cassettes.</td>
		</tr>
		<tr>
			<td>Chemidoc MP Imager&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>Model number: Universal hood III</td>
			<td>Any imaging system with Stain-Free gel imaging capabilities.</td>
		</tr>
		<tr>
			<td>Criterion blotter</td>
			<td>Bio-Rad Laboratories</td>
			<td>1704070</td>
			<td>Includes ice pack, transfer tray, roller, 2 cassette holders, filter paper, foam pads and lid with cables.</td>
		</tr>
		<tr>
			<td>Criterion Cell (Bio-Rad)</td>
			<td>Bio-Rad Laboratories</td>
			<td>1656001</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL (enhanced chemiluminescence)</td>
			<td>Bio-Rad Laboratories</td>
			<td>1705062</td>
			<td>Product name: Clarity Max Western ECL Substrate.</td>
		</tr>
		<tr>
			<td>Electrophoresis buffer 1x Tris Glycine SDS (TGS)</td>
			<td>Bio-Rad Laboratories</td>
			<td>1610772</td>
			<td>Dilute 10x TGS 1 in 10 with ultra-pure H2O.</td>
		</tr>
		<tr>
			<td>Filter paper, 0.34 mm thick</td>
			<td>Whatmann</td>
			<td>3030917</td>
			<td>Bulk size 3 MM, pack 100 sheets, 315 x 335 mm.</td>
		</tr>
		<tr>
			<td>Fine tissue dissecting forceps</td>
			<td>Dumont</td>
			<td>F6521-1EA</td>
			<td>Jeweller&#8217;s forceps no. 5.</td>
		</tr>
		<tr>
			<td>Flat plastic tray/lid&#160;&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Large enough to place the membrane on. Ensure the surface is completely flat.</td>
		</tr>
		<tr>
			<td>Freeze-drying System</td>
			<td>Labconco</td>
			<td>7750030</td>
			<td>Freezone 4.5 L with glass chamber sample stage.</td>
		</tr>
		<tr>
			<td>Freezer -80 oC&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any freezer with a constant temperature of -80 &#176;C is suitable.</td>
		</tr>
		<tr>
			<td>Gel releasers</td>
			<td>1653320</td>
			<td>Bio-Rad</td>
			<td>Slide under the membrane to gather or move the membrane.</td>
		</tr>
		<tr>
			<td>Grey lead pencil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Image lab software&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>N/A</td>
			<td>Figures refers to software version 5.2.1 but other versions can used.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Bio-Rad Laboratories</td>
			<td>1660521</td>
			<td>Any incubator that can be set to 37 &#176;C would suffice.</td>
		</tr>
		<tr>
			<td>Lamp</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer with flea</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane roller&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>1651279</td>
			<td>Can be purchased in the Transfer bundle pack. However, if this product is not available, any smooth surface cylindrical tube long enough to roll over the membrane would suffice.&#160;</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes&#160; (0.6 mL)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG HRP secondary</td>
			<td>Thermo Fisher Scientific</td>
			<td>31430</td>
			<td>Goat anti-Mouse IgG (H+L), RRID AB_228341. Dilute at 1 in 20,000 in blocking buffer.</td>
		</tr>
		<tr>
			<td>Mouse IgM HRP secondary</td>
			<td>Abcam</td>
			<td>ab97230</td>
			<td>Goat Anti-Mouse IgM mu chain. Use at the same dilution as mouse IgG.</td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain I (MHCI) primary antibody</td>
			<td>DSHB</td>
			<td>A4.840&#160;</td>
			<td>Dilution range: 1 in 200 to 1 in 500 in BSA buffer.</td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain IIa (MHCIIa) primary antibody</td>
			<td>DSHB</td>
			<td>A4.74&#160;</td>
			<td>Dilution range: 1 in 200 to 1 in 500 in BSA buffer.</td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain IIx (MHCIIx) primary antibody</td>
			<td>DSHB</td>
			<td>6H1</td>
			<td>Dilution range: 1 in 200 to 1 in 500 in BSA buffer.</td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane 0.45 &#181;m&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>1620115</td>
			<td>For Western blotting.</td>
		</tr>
		<tr>
			<td>Petri dish lid</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tweezers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Pack&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>164-5050</td>
			<td>Product name: Basic power supply.</td>
		</tr>
		<tr>
			<td>Protein ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>26616</td>
			<td>PageRuler Prestained Protein Ladder, 10 to 180 kDa.</td>
		</tr>
		<tr>
			<td>PVDF Membrane 0.2 &#181;m</td>
			<td>Bio-Rad Laboratories</td>
			<td>1620177</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit HRP secondary</td>
			<td>Thermo Fisher Scientific</td>
			<td>31460</td>
			<td>Goat anti-Rabbit IgG (H+L), RRID AB_228341. Dilution same as mouse secondary antibodies.</td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Motic</td>
			<td>SMZ-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Stripping buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>21059</td>
			<td>Product name: Restore Western Blot Stripping Buffer.</td>
		</tr>
		<tr>
			<td>Tissue (lint free)</td>
			<td>Kimberly-Clark professional</td>
			<td>34120</td>
			<td>Product name: Kimwipe.</td>
		</tr>
		<tr>
			<td>Transfer buffer (1x Tris Glycine buffer (TG), 20% Methanol)</td>
			<td>TG: Bio-Rad Laboratories&#160;&#160; Methanol: Merck</td>
			<td>TG buffer: 1610771&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Methanol: 1.06018</td>
			<td>dilute 10x TG buffer with ultra-pure H2O to 1x. Add 100% Methanol to a final concentration of 20% Methanol. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Transfer tray</td>
			<td>Bio-Rad Laboratories</td>
			<td>1704089</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;UV-activation precast gel</td>
			<td>Bio-Rad Laboratories</td>
			<td>5678085</td>
			<td>Gel type: 4&#8211;15% Criterion TGX Stain-Free Protein Gel, 26 well, 15 &#181;L.</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash buffer (1x TBST)</td>
			<td>10x TBS: Astral Scientific&#160; Tween 20: Sigma</td>
			<td>&#160;BIOA0027-4L</td>
			<td>1x TBST recipe: 10x Tris-buffered saline (TBS) is diluted down to 1x with ultra-pure H2O, Tween 20 is added to a final concentration of 0.1%. Store buffer at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Wash containers</td>
			<td>Sistema</td>
			<td>Store bought</td>
			<td>Any tupperware container, that suits the approximate dimensions of the membrane would suffice.</td>
		</tr>
	</tbody>
</table>

fiber type identification of human skeletal muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Identification of individual MHCI, MHCIIa, and MHCIIx muscle fibers using dot blotting 
A feature of MyDoBID is the categorization of the varying MHC and Actin signal intensity strength in a given fiber (Figure 4A). Fiber type was identified by the presence or absence of MHCI and IIa isoforms (Figure 4B). Six fibers showed no MHC or actin detection, indicating that there was no fiber collected. The results of this specific dot blot were the...

Watch this Scientific Journal Video about Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique at JoVE.com

Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

fiber-type-identification-of-human-skeletal-muscle

Research

JoVE Journal

Biochemistry

2.4K Views.  La Trobe University. This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

Video: Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Fiber Type Identification of Human Skeletal Muscle

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An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

Identification of Fatty Acids in Bacillus cereus

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Identification of Functional Protein Regions Through Chimeric Protein Construction

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta