Characterization of Cytoskeletal Proteins

9 April 2019

Jeff White

Darius Artis

Stephon Scott

Morehouse College
 Abstract

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is utilized for the

separation of proteins within biochemical structures based upon their respective molecular

weights; broadly, the migration of the proteinaceous particles through the gel matrix due to size,

wherein smaller molecules will travel faster in the electrical field, is a form of 1-dimensional

analysis. The objective to be derived from this technique was the discernment of the density and

location of proteins in rat heart (H), brain (B), kidney (K), liver extracts (L), as well as a

selection of actin/myosin (A/M). The separated proteins were then stained with Coomassie Blue,

with the surfeit of dye having been eluted through a de-staining treatment, in order to facilitate

the visualization of the Coomassie dye ligands that have bound to the side chains of the

examined polypeptides via weak chemical interactions; this quantification of the proteins in the

polyacrylamide gel medium evidenced itself as dark bands in each lane that contained a rodent

tissue relative to the molecular weight marker (MW) that ranged from 10 kilodaltons to 250

kilodaltons (kDa). Adjacent to the molecular weight marker in the second lane were known

samples of actin/myosin; it was surmised that the prominent band at the 200 kilodalton marker

was representative of myosin and another band around the 37 kilodalton marker was indicative

of an abundance of the protein actin. The determined molecular weights of the known protein

samples were verified through the consultation of external research articles. In addition to the gel

with the general protein stain, a second gel was prepared for the process of Western blot (WB)

assay which was effectuated by the transference of the proteins being examined into a

nitrocellulose membrane. This variant of polypeptide immunoblotting of fractions of tissue

homogenates was implemented in order to specifically detect myosin in the samples; this was

done through the implementation of a murine-derived primary antibody, 1% anti-myosin (HC),

and subsequent application of a secondary, goat anti-mouse antibody. Chemiluminescence was

employed to qualitatively illustrate the indirect recognition of the target protein myosin. It was

hypothesized by the laboratory coterie that the heart would be comprised of the most abundant

protein band corresponding to the molecular weights of actin and myosin due to its role as a

muscular pump. This assertion was verified upon inspection of the sixth lane of the Coomassie-

stained gel as it was typified by several prominent bands, particularly between the 37 kDa and 50

kDa as well as the 200 kDa markers, whereas the other lanes contained noticeably dimmer

protein band expressions; this is significant as there is a direct correlation between the

comparative intensity of a background band of a Coomassie signal and protein concentration.

Furthermore, the accuracy of the predictive statement that the Western blot evaluation would

show that the tissue samples would all contain myosin, because of the nigh ubiquitous presence

of its isoforms in a myriad of eukaryotic cells, could not be assessed as the visual data from the

corresponding blotting matrix was unfit for interpretation.

Introduction

With respect to the analysis of proteins, polyacrylamide gel electrophoresis is a frequently used

technique to carry out those ends. Its proper application is predicated on the dissemination of

electrically-charged particles via an anode through a matrix of polyacrylamide gel wherein ions

from dissociated salts are used to conduct the electrical current; moreover, this is concomitant

with the introduction of the detergent sodium dodecyl sulfate or SDS. SDS is an anionic, water-

soluble agent that acts as a denaturant, much like urea or β-mercaptoethanol, by disrupting the

chemical forces that stabilize the tertiary or secondary confirmations of globular polypeptides as

well as enabling easier access to the intracellular environ by altering the structural integrity of
the lipids in the membrane bi-layers of the cells (Nowakowski, 2014). In uniformly mantling the

proteins with the surfactant, a detergent-protein complex is formed which leads to the covering

of the intrinsic charges of the relevant proteins and confers them with an analogous charge to

mass proportion; this occurs due to the loss of the functional structure of the proteins resultant

from the procedure of denaturation, all that remains is the sequence of amino acids or primary

structure, which informs the rod-like alignment and negative charge that all of the proteinaceous

complexes adopt (Nowakowski, 2014). Thus, the vector affecting the movement of the protein

fractions through the discontinuous, polyacrylamide matrix is size rather than charge or shape.

Prior research abjectly concerning the SDS-PAGE technique has been centered on the

assessment of proteinuria, or inordinate amounts of chains of amino acid residues in the urine, as

a component of HIV testing (Nowakowski, 2014). In this experiment, visual data from the

proteins that were separated was documented through protein staining with Coomassie Blue dye

and use of Western blot methodology.

Western Blotting allowed for the detection of the motor protein myosin in the separated proteins

from the rat tissue extracts. The interpretation of which was predicated upon the successful

transference of the proteins from the polyacrylamide medium, which was sandwiched between a

nitrocellulose membrane and filter paper, and the administration of a primary as well as a

secondary antibody. A perpendicular electrical current, executed through attached electrodes at a

90-degree angle, was conducted through the gel-membrane assemblage and elicited the

relocation of the negatively charged proteins from the polyacrylamide gel in a buffer solution to

the positive charge that was induced in the nitrocellulose membrane which also has a high

affinity for the proteins (Luo, 2011). The completion of this sequence lead into the specific

targeting of myosin in the samples through antibody applications. The carrying out of this SDS-
PAGE technique in its entirety was what assisted in the actualization of the purpose of this

experiment which was to characterize the purified protein bands of the rodent-sourced tissue by

comparing their sizes to the molecular weights of the known samples. The hypothesis being

tested was that if the polyacrylamide gel was treated with the Coomassie Blue stain, then it

would show that the heart tissue would have the highest concentration of actin and myosin. An

additional predicted outcome was that the Western blot analysis would reveal that the rat brain,

kidney, heart, and liver would all exhibit the presence of myosin.

Methods

Ancillary materials were initially collected at the laboratory benches to include the actin/myosin

(AM), brain extract (B), kidney extract (K), heart extract (H), and liver extract (L) protein

samples, a molecular weight marker (MW) to provide a frame of reference for the relative

masses of the proteinaceous fractions, and a pre-cast polyacrylamide gel. Next, the tissue

samples were allotted time to thaw while 1L of a 1X Tris-Glycine-SDS (TGS) Gel Running

Buffer was prepared from a 10x stock solution; the satisfactory ratio of solute to solvent was

ascertained by algebraically manipulating the dilution equation C1V1= C2V2 to yield 900 mL of

H2O to 100 mL of the stock 10X TGS buffer solution. The Tetra Gel Box was then set-up

according to the prescribed specifications and the working 1L 1X solution was added to it; the

buffer was used to resist any changes to the pH of the medium. The polyacrylamide gel cassette

was prepared by removing the comb from it and putting it into the conductor system while the

shorter plate of the cassette was turned towards its inner regions which forms part of the inner

chamber assembly. This apparatus is then set inside of the Tetra Gel Box whose tank was

completely filled with the working 1X solution until the TGS buffer was visible just past the base
of the gel plates. The gels were then loaded using a p200 pipet after a guide was affixed to the

top of the electrode apparatus in accordance with the following table:

Lane Sample Volume (μL)

1 Molecular Weight 15
2 Actin/Myosin (AM) 30
4 Brain 30
6 Heart 30
8 Kidney 30
10 Liver 30

It should be noted the pipet did not touch the bottom of the well, lest the gel is compromised, and

remained in front of the well number during dispensation of the samples. Thereupon, the gel was

electrophoresed for 30 minutes at 200 volts, removed from the cassette that encased it, and

placed in a petri dish with 15 mL of the dye Coomassie Blue to incubate for 15 minutes. It was

then washed with dH2O after the excess stain was poured off and 15 mL of de-stain solution was

added to the dish. The de-stain process occurred over the course of a week so that the gel could

later be scanned to produce images of the separated proteins.

The subsequent week, the proteins were transferred into a different substance; a polyacrylamide

gel and nitrocellulose membrane were placed between two layers of filter paper and sponge with

a current running through them for 15 minutes at 100 voltage. After the medium transference, a

block was added (0.05% milk in 1X PBS) before a 10-minute incubation period with the primary

antibody, made in mice, 1% antibody anti-myosin (HC). The remainder of the antibody was

washed off and the secondary antibody, goat anti-mouse antibody, was used with an additional

10-minute incubation period. Developer reagents were put in after the excess secondary antibody

was washed off; the laboratory groups then waited for 5 minutes. While a protein immunoblot
will typically take at least an additional week, the efficacy of the equipment in the laboratory

allowed the respective groups to expedite that portion of the protein fingerprinting to finish it

within the same day. With the Western blotting procedure completed, the nitrocellulose gel was

ready for visualization.

Results

Figure 1. Examples of SDS-PAGE gels of Murine Tissue Extracts. Separation of protein

fractions was facilitated by gel electrophoresis with the synthesis of visual data being
synthesized through the implementation of either a general protein stain consisting of the dye
Coomassie Blue (A) or Western blotting (B) with the mouse, monoclonal anti-myosin antibody
to the myosin-common site-chain.
A. B.

Figure 2. Calibrated Unstained Protein Standard Reference. An image containing molecular

weight markers ranging from 10-250 kDa for the purposes of the classification of the sizes of
electrophoresed, proteinaceous fragments in Western blots and SDS-polyacrylamide gels (A).
A.
Image (A) of Figure 1 illustrates electrophoresis in 1-dimension, or separation of proteinaceous

selections by size, with the smaller fractions at the bottom of the gel having migrated through the

matrix away from the negatively-charged cathode at the top at a faster rate than the larger

particles. In Lane 1 is the molecular weight marker, or protein ladder, which is a standard that is

used to approximate the size of molecules in electrophoresed gel based upon the inverse

relationship between molecular weight and the distance traveled towards the anode

(Nowakowski, 2014). The placement of the protein bands in Lane 1 were compared to the

resolved bands in a unstained ladder, as seen in Figure 2, in order to develop a reference by

which the sizes of the fractions in the other lanes could be approximated. Lane 2 consists of the

actin/myosin (AM) control which allows the bands evident in the tissue extracts to be

characterized based upon the size of this known sample; the most abundant proteins in this lane

were at the 200 kDa marker and between the 37 and 50 kDa markers which were established as

myosin and actin. This was confirmed as the molecular subunits of the motor protein myosin

have an estimated molecular weight of 215,000 Daltons, or 215 kDa, and the molecular weight

of actin is cited as 42,000 Daltons, or 42 kDa (Müller, 2013). In Lane 4 is the rat brain sample
which has a prominent band at what would be the 200 kDa marker that parallels the same band in

the AM control; this is indicative of the presence of a heavier protein such as myosin which is

comprised of heavy chains of roughly 215 kDa and is instrumental in a multitude of motility

activities in eukaryotic cells (Müller, 2013). Lane 6, the heart extract, has the most intense or

darkest stain of all of the specimens that were examined. This demonstrates that the heart sample

has the highest concentration of proteins in comparison to the other specimens. Its most

prominent bands are localized at the 200 kDa marker, which is most likely a heavy chain of

myosin, and between the 37kDa and 50 kDa markers. Given that the molecular weight of actin is

of the order of 42 kDa, which is confirmed by a similar band in the AM control lane, it can be

reasonably asserted that this data depicts the presence of actin in the tissue considering these

monomeric subunits are a constituent of the contractile network in fibrous tissues (Müller, 2013).

The rat-derived kidney extract is in Lane 8 of Image (A). The protein band in this lane with the

highest visibility is towards the bottom of the molecular weight standard, suggesting a protein of

smaller size, at around the 25 kDa marker. This could signify the presence of the enzyme

carbonic anhydrase, which has a molecular weight of 30 kDa and in the kidney its regulation of

bicarbonate ions influences pH and fluid content (Pinard, 2015). Lane 10 contains the liver

extract which appears to have a dark band at the 100 kDa marker. This could relate to the

monomer glycogen phosphorylase which has a molecular weight of 97.34 kDa and is

predominant in adult, mammalian livers as a locus of glucose production (Nagy, 2017).

Image (B), which concerns Western blotting with an antibody, should show the transferred

protein bands as they pertain to the prevalence, or lack thereof, of the target protein; the applied

1X PBS block should have reduced the incidence of any protein interactions between the

nitrocellulose medium and antibody that were non-specific (Luo, 2011). Due to an undetermined
error in the blotting process, the visualization of the second gel by chemiluminescence was

empirically inconsequential thereby removing the practical relevance from any attempts at

extrapolation.

Discussion

The primary objective of this lab was to determine the most abundant protein in the rat brain,

heart, kidney, and liver samples in the gel with the general protein stain; the sizes of the diffused

fractions were compared to the known values in the actin/myosin control and conclusion were

drawn from there. Additionally, the transference of the protein bands from the polyacrylamide

gel to the nitrocellulose membrane through the Western blot procedure, which was then treated

with an antibody, was to be examined for the presence of the protein myosin. The hypothesis that

the members of the laboratory group devised before conducting the experiment was that if the

first gel was stained with the dye Coomassie Blue, then it would show that the heart sample

would contain the highest concentration of actin and myosin. Even in light of this it was

predicted that the Western blot would elucidate that the motor protein myosin would be present

in all of the provided tissue specimens. The major findings from the collected data were a series

of prominent protein bands in each of the samples which are as follows: the preponderance of

proteins were at the 200 kDa marker in the brain tissue, at the 200 and 40 kDa markers in the

heart extract, at the 25 kDa marker in the kidney sample, and at the 100 kDa marker in the lane

containing the liver extract. The visual data of the Coomassie-stained gel confirmed the

hypothesis of the group as it was resolved that the heart tissue did contain the highest

concentration of actin and myosin after comparing the size of its most visible bands to those in

the known samples; although other extracts, such as the brain, contained similar protein bands
their stain was incontrovertibly dimmer which is indicative of a lower density as the relative

intensity of the stain itself is a function of protein concentration. It is logically sound that the

heart would contain more actin and myosin than another organ like the kidney as it is essentially

a muscular pump that converts chemical energy into mechanical energy that manifests as

contractions which help circulate blood through the body; actin filaments in conjunction with

myosin are responsible for the generation and constancy of this type of motility (Müller, 2013).

The predictive statement that the Western blot would show that myosin was present in all of the

samples could not be investigated as the visual data for the second gel contained protein bands

that were not arranged in a linear fashion as was expected for the purposes of interpretation.

Sources of this peculiarity could be attributed to the sub-optimal dispensation of the samples,

inadequate development as a result of a contaminated reagent, or human error during the

transference step like not calibrating machinery for the correct voltage (Gilda, 2015).

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