Creating Optimal Western Blot Conditions for OPA1 Isoforms in Skeletal Muscle Cells and Tissue

Abstract

OPA1 is a dynamin‐related GTPase that modulates mitochondrial dynamics and cristae integrity. Humans carry eight different isoforms of OPA1 and mice carry five, all of which are expressed as short‐ or long‐form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial energetics and DNA maintenance. However, western blot isolation of all long and short isoforms of OPA1 can be difficult. To address this issue, we developed an optimized western blot protocol based on improving running time to isolate five different isoforms of OPA1 in mouse cells and tissues. This protocol can be applied to study changes in mitochondrial structure and function. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Western Blot Protocol for Isolating OPA1 Isoforms in Mouse Primary Skeletal Muscle Cells

INTRODUCTION

Optic atrophy‐1 (OPA1) is a GTPase that localizes to the inner mitochondrial membrane and is a central player in mitochondrial fusion (Chen et al., 2009). Mitochondrial fusion is essential for maintaining mitochondrial network morphology and function. OPA1 also regulates cristae organization, protecting against apoptosis (Frezza et al., 2006), and maintaining respiratory chain assembly and functionality, mitochondrial membrane potential, mitochondrial calcium homeostasis, and mitochondrial DNA (Belenguer & Pellegrini, 2013; Chen et al., 2009). Variants in OPA1 have been linked to a wide range of neurodegenerative diseases, including autosomal dominant optic atrophy and Behr syndrome (Alexander et al., 2000; Delettre et al., 2000). Further research on OPA1 and its functions may lead to new treatments for various diseases.

While humans express eight OPA1 mRNA transcripts by alternate splicing of exons 4, 4b, and 5b (del Dotto et al., 2017), mice express four splice variants, 1, 5, 7, and 8, through alternate splicing of only exons 4b and 5b (Akepati et al., 2008) (Fig. 1). Murine isoforms can present as long‐ or short‐form isoforms, which are associated with different mitochondrial functions (Figure 1). Isoforms 5 and 8, due to the presence of an S3 cleavage site encoded by the 4b exon, are constitutively cleaved by the protease YME1L and are expressed exclusively as short‐form isoforms (Wang et al., 2021). Isoforms 1 and 7 lack the S3 site, so they are expressed as either a long‐ or short‐form isoforms. The relative level of short‐ or long‐form expression is regulated by several proteases, which are responsible for processing OPA1. For instance, YME1L will constitutively cleaves long‐form isoforms 7 at the S2 domain, encoded by the 5b exon, to produce short‐form isoforms. However, cleavage of OPA1 by other proteases can be triggered under stressful conditions. For example, when membrane potential decreases, the metalloprotease OMA1 will cleave long‐form isoforms 1 and 7 into short‐form isoforms via cleavage of the S1 domain, increasing fission (Gilkerson et al., 2021). Cleavage of isoforms 1 and 5 will produce the same short‐form isoforms when isoform 1 is fully processed by the moderate action of the proteases, which will take longer due to the lack of the S3 site. Isoforms 7 and 8 will produce the same two short isoforms when their long form (l‐OPA1) is cleaved at the S1 domain by OMA1 or the S2 domain by YEM1L. Isoforms 5 and 8 are exclusively processed into short forms (Akepati et al., 2008). A recent study described a third cleavage site for OPA1, site 3, encoded by the 3′ half of exon 4B and cleaved by YME1L (Wang et al., 2021). Long‐ and short‐form isoforms play different roles in restoring cristae structure, mitochondrial DNA (mtDNA) abundance, and energetic efficiency (del Dotto et al., 2017). Certain experimental conditions, typically associated with cellular stresses such as cold exposure, cause increased cleavages of OPA1 into short isoforms (Pereira et al., 2021). However, short‐form isoforms still play important roles in energetic efficiency, which requires a balance between short and long isoforms (del Dotto et al., 2017). Recently, researchers found that OPA1‐specific isoforms may have specific functional roles. For instance, 1 and 7 have therapeutic potential and are implicated in diseases caused by mitochondrial dysfunction (Maloney et al., 2020). Therefore, it is important to efficiently isolate and study different OPA1 isoforms to understand how they work and how their collective and differentiated functions maintain cellular health under various conditions. In this Basic Protocol, we describe the optimal conditions for isolating OPA1 isoforms by western blotting.

Figure 1.

Schematic representation of the four murine OPA1 splice variants and the six distinctive bands (labeled in parentheses (A‐E)). The mouse Opa1 gene has four splice variants, isoforms 1, 7, 5, and 8. Isoforms 5 and 8 contain an S3 cleavage site, which is constitutively cleaved by YME1L to produce the short‐form (s‐OPA1) variant. Isoforms 1 and 7 are initially expressed as the long‐form (l‐OPA1) variant, but cleavage at S1, by OMA1, or S2, by YEM1L, converts them to the s‐OPA1 variant. The s‐OPA1 variant of isoform 1 is identical to the s‐OPA1 isoform 5. The two s‐OPA1 variants of isoform 7 are identical to the s‐OPA1 variants of isoform 8. MTS, mitochondrial targeting sequence; TM, Transmembrane domain; CC‐1, Coiled‐coil domain; MPP, mitochondrial processing peptidase, domains 4, 4b, 5, and 5b are encoded by their respective exons 4, 4b, 5, and 5b. Arrows on the left denote the alternate splicing from the splice variants to lower molecular weight forms.

NOTE: All protocols involving animals must be reviewed and approved by the appropriate Animal Care and Use Committee and must follow regulations for the care and use of laboratory animals. Appropriate informed consent is necessary for obtaining and use of human study material.

WESTERN BLOT PROTOCOL FOR ISOLATING OPA1 ISOFORMS IN MOUSE PRIMARY SKELETAL MUSCLE CELLS

In this protocol, samples for western blot analysis are isolated from lysed mouse primary skeletal muscle cells, loaded onto a gel, and run using optimized conditions to provide high‐quality isolation of OPA1 isoforms. Samples are transferred to a membrane for incubation and protein detection using BD Biosciences OPA1 antibody.

Materials

• Preferred cell line (e.g., Primary murine myoblasts)

• Anti‐OPA1 antibody (BD Biosciences, cat. no. 612606)

• NaCl (ThermoFisher, cat. no. 7647‐14‐5)

• Tris base (Millipore Sigma, cat. no. 77‐86‐1)

• SDS (Millipore Sigma, cat. no. 151‐21‐3

• Triton X‐100 (ThermoFisher, cat. no. 9002‐93‐1)

• Sodium deoxycholate monohydrate (ThermoFisher, cat. no. 145224‐92‐6)

• EDTA (Millipore Sigma, cat. no. 60‐00‐4)

• Protease inhibitor tablet (Roche, cat. no. COEDTAF‐RO)

• Glycerol (Millipore Sigma, cat. no. 56‐81‐5)

• β‐mercaptoethanol (Millipore Sigma, cat. no. 60‐24‐2)

• Bromophenol blue (ThermoFisher, cat. no. 115‐39‐9)

• DTT (ThermoFisher, cat. no. R0861)

• Glycine (Millipore Sigma, cat. no. 56‐40‐6)

• Methanol (Millipore Sigma, cat. no. 67‐56‐1)

• Tween‐20 (Millipore Sigma, cat. no. 9005‐64‐5)

• Dulbecco's Modified Eagle Medium (DMEM) (ThermoFisher, cat. no. 11965084)

• Fetal bovine serum (FBS) (ThermoFisher, cat. no. 16000044)

• PBS (ThermoFisher, cat. no. 10010023)

• 100× Penicillin/streptomycin (Pen/Strep) (ThermoFisher, cat. no. 10378016)

• BSA (Millipore Sigma, cat. no. 9048‐46‐8)

• TBS (Bio‐Rad, cat. no. 1706435)

• 2× Laemmli sample buffer (Bio‐Rad, cat. no. 1610737)

• SuperSignal^™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher, cat. no. 34579)

• 100‐mm plate (Corning, cat. no. 353003)

• Microcentrifuge tubes (e.g., ThermoFisher, cat. no. AM12450)

• 22‐G needle (e.g., Amazon, cat. no. LY‐999)

• 4%–20% Tris‐glycine Mini Gels (Novex, cat. no. XP04205BOX)

• Vertical electrophoresis system (Bio‐Rad, cat. no. 1658004)

• Nitrocellulose membrane (Bio‐Rad, cat. no. 1620115)

• Microcentrifuge (e.g., Eppendorf Digital Centrifuge, cat. no. 5415D)

• Invitrogen iBright FL1500 (Invitrogen, cat. no. A44241)

• GraphPad (www.graphpad.com)

• Image J (https://imagej.net/)

Day 1

CRITICAL : All media and reagents must be chilled in advance, preferably in a 4°C fridge for at least 24 hr.

This section describes how to collect and prepare samples.

• 1Prior to beginning, use standard and validated protocols (such as Hindi et al., 2017; Shahini et al., 2018; Stephens et al., 2023) for the isolation, culturing, and differ‐ entiation of primary murine myoblasts, or other preferred cell line. Grow cells to an appropriate confluency on a 100‐mm plate in standard DMEM or as described in the relevant culture protocol.
• 2Aspirate media
• 3Wash cells with ∼10 ml ice‐cold PBS to remove residual media
• 4Immediately aspirate PBS
• 5Add pre‐chilled RIPA buffer directly to cells.
• 6Immediately scrape cells from the plate
• 7Transfer cells to a microcentrifuge tube.

  Use approximately 1 ml RIPA buffer per 100‐mm plate. Scale up or down as necessary.

• 8Incubate the microcentrifuge tubes on ice for 10 min (gently vortex every 2–3 min)

  Lysates can also be passed through a 22‐G needle to aid in solubilization.

• 9Centrifuge the microcentrifuge tubes at 7245 RCF for 15 min.

  This section describes how to run protein samples on an SDS‐PAGE gel

• 10Load 20–50 µg of lysate with loading dye (Bio‐Rad 2X Laemmli sample buffer and add BME)
• 11Run lysate samples on Novex 4%–20% Tris‐glycine Mini Gels (pre‐chilled for 1 hr in the cold running buffer before use).

  Gels may also be custom‐made, but it is important to use a gradient gel, as they provide better separation than a normal gel for the close molecular weights of OPA1 isoforms.

  It is important to ensure that all materials and reagents, especially the Novex Mini Gels, are chilled for an hour before use.

• 12Run the gel for 10 min at 100 V (small gel boxes) to get the sample through the wells.

  For larger gel boxes, 170–200 V can be used, but multiple isoforms may not be seen.

• 13Reduce the voltage to 50 V for 5–7 hr at 4°C.

  Gels can be run overnight at 35 V and 4°C.

Day 2

This section describes how to transfer proteins from a gel to a membrane.

• 14Transfer the Tris‐glycine gel to a nitrocellulose membrane for 12 hr at 35 mV in a 4°C cold room.

Antibody Incubations

• 15Wash the membrane in 1× TBS for 5 min.
• 16Submerge the membrane in blocking buffer.
• 17Shake membrane in blocking buffer overnight at 4℃ or 1 hr at room temperature.

Day 3

• 18Wash the membrane three times with TBST for 5 min each.

  It is important to switch from TBS to TBST, as the additional Tween‐20 is necessary to wash away weaker non‐covalent and hydrophobic interactions.

• 19Incubate membrane with primary antibody for 1 hr at room temperature.

  A 1:1000 primary antibody dilution, per the manufacturer's suggestion, is recommended. Subsequent experiments may be further optimized depending on the results.

• 20Wash three times with TBST for 5 min each.
• 21Incubate and shake the membrane with the appropriate horseradish peroxidase‐conjugated secondary antibody for 1 hr at room temperature.
• 22Wash the membrane three times with TBST for 5 min each.

Protein Detection

• 23Prepare ECL detection reagent by mixing solutions A & B in a 1:1 ratio.
• 24Cover the membrane with ECL solution
• 25Incubate for 5 min at room temperature with gentle rocking.
• 26Remove the membrane from the solution.
• 27Wrap the membrane in plastic wrap, while eliminating air bubbles.
• 28Place the membrane in an exposure cassette.
• 29Expose to film for different amounts of time.
• 30Read using the Invitrogen iBright FL1500 for imaging and quantification.

REAGENTS AND SOLUTIONS

Buffers

To lyse the cells, we used a RIPA buffer:

Reagent	Stock concentration	Final concentration	Amount
NaCl	5 M	150 mM	320 µl
Tris pH 7.2	1.0 M	10 mM	100 µl
SDS	10%	0.1%	100 µl
Triton X‐100	100%	1.0%	100 µl
deoxycholate	10%	1%	80 µl
EDTA	0.5M	5 mM	40 µl
Di H2O	N/A	N/A	9.26 ml
Protease inhibitor tablet	N/A	N/A	1 Tablet
Total	N/A	N/A	10 ml

Once cell lysate samples are obtained, we mixed our samples with 6× sample buffer before loading:

Reagent	Stock concentration	Final concentration	Amount
Tris pH 6.8	1.5 M	0.375 M	2.5 ml
Glycerol	100%	48%	4.8 ml
SDS	N/A	6%	0.6 g
β‐mercaptoethanol	100%	6%	0.6 ml
Bromophenol blue	N/A	0.06%	0.006 g
DTT	N/A	0.6 M	0.930 g
DI H2O	N/A	N/A	2.1 ml
Total	N/A	N/A	10 ml

After loading samples onto an SDS‐PAGE gel, we used 1× Tris‐glycine running buffer

Reagent	Stock concentration	Final concentration	Amount
Tris base	N/A	25 mM	12.1 g
Glycine	N/A	250 mM	75.06 g
SDS	N/A	0.1%	4 g
DI H2O	N/A	N/A	4 L

To transfer to a membrane, we used a 1× Tris‐glycine transfer buffer:

Reagent	Stock concentration	Final concentration	Amount
Tris base	N/A	25 mM	12.1 g
Glycine	N/A	192 mM	57.6 g
Methanol	100%	20%	800 ml
SDS	N/A	0.02%	0.8 g
DI H2O	N/A	N/A	4 L

The following buffers are needed for antibody incubations.

Blocking buffer:

Reagent	Stock concentration	Final concentration	Amount
TBS	10×	1×	10 ml
Tween‐20	100%	0.1%	0.1 ml
BSA	N/A	5%	5 g
DI H2O	N/A	N/A	89.9 ml
Total	N/A	N/A	100 ml

Antibody dilution buffer:

Reagent	Stock concentration	Final concentration	Amount
TBS	10×	1×	10 ml
Tween‐20	100%	0.1%	0.1 ml
BSA	N/A	5%	5 g
DI H2O	N/A	N/A	89.9 ml
Total	N/A	N/A	100 ml

Wash buffer TBST:

Reagent	Stock concentration	Final concentration	Amount
TBS	10×	1×	10 ml
Tween‐20	100%	0.1%	0.1 ml
DI H2O	N/A	N/A	89.9 ml
Total	N/A	N/A	100 ml

COMMENTARY

Critical Parameters

This protocol is designed to make use of OPA1 BD monoclonal antibody and extended western blot run times. Deviation from these key parameters may cause fundamental issues in this protocol.

Troubleshooting Table

 

Problem	Possible cause	Solution
Did not detect any bands on your membrane.	Retention time issues or expired antibodies.	There are several ways to increase the retention time. Increase the primary antibody inoculation time to O/N rocking in a cold room. Alternatively, increase the secondary antibody to no longer than 2 hr or increase the film exposure time. This lack of detection could result from the antibody not having enough time to bind to the proteins on the membrane. Also, we recommend being sure that the primary antibody has not expired, as that would decrease its effectiveness.
Protein did not transfer from the gel to the membrane.	Unoptimized protein or incorrect loading order	This can be checked by staining the gel with Ponceau S and Coomassie Brilliant Blue. Ponceau S is less sensitive but reversible. If bands show up then the proteins were not transferred. It is also important to ensure that the stacking order is correct to avoid issues. Finally, it is important to optimize transfer to avoid weak or undetectable signals without over‐transferring, which can lead to nonspecific binding.
Unable to tell how many isoforms are on the band	Running time too fast	Smaller separation means a smaller distribution of the different isoforms. We have found that 5–7 hr separation at 50 V is key to discerning 4–5 isoforms. However, increased retention time may be necessary.

Statistical Analysis

Bands from the blot are quantified using ImageJ. Statistical analyses are performed using one‐way ANOVA on GraphPad software, and significant difference is defined as P < 0.05.

Understanding Results

Although OPA1 deficiency causes a loss of mitochondrial morphology, it also promotes insulin sensitivity in a fibroblast growth factor 21‐dependent manner (Pereira et al., 2017). A prior study in cardiomyocytes showed that 2 isoform bands of OPA1 and 5 isoforms of OPA1 showed similar changes in expression after insulin stimulation (Parra et al., 2013). Given that the AKT pathway aids in regulating Opa1 cleavage (Cai et al., 2019), it is reasonable to expect concomitant changes in AKT and the active phosphorylated form of AKT. Furthermore, insulin stimulation increases AKT expression (Kim et al., 1999). Notably, recent studies in cardiomyocytes showed that insulin stimulation activates the Akt‐mTOR‐NFκB pathway to increase OPA1 (Parra et al., 2013). To investigate this paradigm, we used C2C12 myotubes and, following serum starvation overnight, performed insulin treatment (10 nM/L) in 2‐hr increments. To begin, we performed the above western blot procedure with OPA1 BD monoclonal antibody (Fig. 2A), which showed a significant increase of OPA1 levels at 2 hr and 4 hr after insulin stimulation in C2C12 (Fig. 2B). Here, we show that changes in the total expression of OPA1 can be observed by looking at 5 isoforms of OPA1. Paralleling this was a similar change in AKT across 6 hr of insulin stimulation (Fig. 2C and 2D). We saw similar increases in AKT and OPA1, marked by significant progressive increases after 2 hr followed by a decrease from 4 hr and 6 hr (Fig. 2C and 2D). We used two factors to allow for normalization and as an internal control gene, GAPDH and alpha‐tubulin. Together, these results confirm our recent findings of elevated OPA1 following insulin stimulation in primary myoblasts and myotubes (Stephens et al., 2023).

Figure 2.

Example of five OPA1 bands with BD monoclonal anti‐OPA1 mouse antibody. Post 4‐hr insulin stimulation, increased OPA1 levels were observed in C2C12 myotubes. (A) Western blot expression of OPA1 and alpha‐tubulin, following treatment with insulin for 0–6 hr. (B) OPA1 levels normalized to alpha‐tubulin expression across insulin stimulation intervals. (C) Western blot of AKT/Protein Kinase B and phosphorylated AKT, the active form of AKT, following treatment with insulin for 0–6 hr. (D) pAKT levels over total AKT protein normalized to GAPDH expression across insulin stimulation intervals. N = 6 per treatment in triplicate; ** indicates p‐value < 0.01, *** indicates p‐value < 0.001.

We validated this approach by visualizing 5 isoform bands in various murine skeletal muscle tissue types using OPA1 BD monoclonal antibody, with alpha‐tubulin as a loading control. We observed five bands in the murine gastrocnemius muscle (Fig. 3A) and then observed the same five bands in murine soleus tissue (Fig. 3B). This suggests the western blot technique can be applied to other tissue types. To confirm the utility of this protocol, we ran a standard western blotting protocol (Mahmood & Yang, 2012) in primary skeletal muscle myoblasts. With the truncated running times of the standard method, only two OPA1 bands were seen (Fig. 4), highlighting the necessity of a modified protocol to maximize OPA1 bands visualized via immunoblotting.

Figure 3.

Example of five OPA1 bands with BD monoclonal anti‐OPA1 mouse antibody. (A) Western blot expression of OPA1 and alpha‐tubulin in murine gastrocnemius muscle. (B) Western blot expression of OPA1 and alpha‐tubulin in soleus muscle

Figure 4.

Examples of two OPA1 bands in primary skeletal muscle myoblast. OPA1, normalized to GAPDH, was compared in an adenovirus with Cre recombinase enzyme (Ad‐Cre) and an adenoviral vector with green fluorescent protein (Ad‐GFP)

Future Perspectives

The main limitation of western blotting is that researchers are confined to using commercially available primary antibodies for detection. Beyond this, cross‐reactivity with other proteins or nonspecific binding can lead to false‐positive or false‐negative results. Here, specific antibodies are utilized depending on the OPA1 isoform desired, but polyclonal antibodies in certain cell types, such as cardiomyocytes, may not detect modifications in the levels of the OPA1 protein at an early stage (Parra et al., 2013). Additionally, the type of gradient gel used (Wang et al., 2021) can improve the visualization of all OPA1 isoforms. It is also important to note that visualization of all expression levels of OPA1 isoforms may be model/tissue/cell‐dependent. We have only noted five protein bands using this protocol, but further optimization with gradient gel can allow visualization of a sixth band due to claevage of S3 isoforms at the S3 domain (del Dotto et al., 2017; Maloney et al., 2020). Specifically, future advances may look at the relative abundance of specific bands, as the top two bands are hypothesized to be long forms of OPA1, while the bottom three bands are thought to be short isoforms (Song et al., 2007). Elucidating the expression of these specific isoforms may be important, given their different roles (del Dotto et al., 2017).

Looking at other proteins, such as AKT, to confirm associated changes may be important. Finally, it is possible that, depending on the experimental conditions, cleavage of OPA1 to C‐terminal fragments is happening, causing bands not to occur (Sood et al., 2014). Given that this C‐cleavage is dependent on Mfn2, and not common cleavage factors like the metalloprotease OMA1, it can be more difficult to look at upstream factors in such scenarios (Gilkerson et al., 2021).

Time Considerations

This protocol is optimized to be performed across three continuous days.

Author Contributions

Margaret Mungai: Conceptualization; writing—original draft; writing—review and editing. Amber Crabtree: Conceptualization; writing—original draft; writing—review and editing. Han Le: Writing—review and editing. Johnathan Moore: Writing—review and editing. Desiree Nguyen: Writing—review and editing. Chanel Harris: Writing—original draft; writing—review and editing. Dominique Stephens: Writing—original draft; writing—review and editing. Heather Beasley: Writing—original draft; writing—review and editing. Edgar Garza Lopez: Writing—original draft; writing—review and editing. Kit Neikirk: Writing—original draft; writing—review and editing. Bryanna Shao: Writing—original draft; writing—review and editing. Ashton Oliver: Writing—review and editing. Genesis Wilson: Writing—original draft; writing—review and editing. Serif Bacevac: Writing—original draft; writing—review and editing. Larry Vang: Writing—original draft; writing—review and editing. Zer Vue: Writing—original draft; writing—review and editing. Neng Vue: Writing—original draft; writing—review and editing. Andrea Marshall: Writing—original draft; writing—review and editing. Kyrin Turner: Writing—original draft; writing—review and editing. Elma Zaganjor: Writing—original draft; writing—review and editing. Jianqiang Shao: Writing—original draft; writing—review and editing. Sandra Murray: Writing—original draft; writing—review and editing. Jennifer Gaddy: Writing—original draft; writing—review and editing. Celestine Wanjalla: Writing—original draft; writing—review and editing. Jamaine Davis: Writing—original draft; writing—review and editing. Steven M. Damo: Writing—original draft; writing—review and editing. Antentor Hinton: Conceptualization; supervision; writing—original draft; writing—review and editing.

Conflict of Interest

The authors declare no conflict of interest.

Mungai, M. , Crabtree, A. , Le, H. , Moore, J. , Nguyen, D. , Rodriguez, B. , Harris, C. , Stephens, D. C. , Beasley, H. K. , Garza‐Lopez, E. , Neikirk, K. , Shao, B. , Oliver, A. , Wilson, G. , Bacevac, S. , Vang, L. , Vue, Z. , Vue, N. , Marshall, A. G. , … Hinton, A. (2025). Creating optimal western blot conditions for OPA1 isoforms in skeletal muscle cells and tissue. Current Protocols, 5, e70004. doi: 10.1002/cpz1.70004

Data Availability Statement

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Antentor Hinton (antentor.o.hinton.jr@Vanderbilt.Edu).

Materials availability

All generated materials, if applicable, are created using the methods highlighted in the text above.

Data and code availability

Full data and requests for data and code availability should be directed to and will be fulfilled by the lead contact, Antentor Hinton (antentor.o.hinton.jr@Vanderbilt.Edu).