APPLICATION NOTE #201508

Protein Extraction from Soft Tissues using
the Fisher Scientific Bead Mill 24
Brandon Easparro

Introduction

Materials & Methods

Proteomic profiling attempts to elucidate
not only protein repertoire, but thestructure and function of proteins. Post
genome sequencing, scientists are now
focusing on studying what proteins aredecoded from the genome. The first stepin many proteomic studies is the extraction
and isolation of proteins from the sampleof interest. The success of a protein assay
is often dependent on quality and methodof this first step. A common method forextraction of proteins from animal tissues ismechanical homogenization. While manytissue types can be robust and requiresignificant mechanical force to dissociatethem and access their cellular components,
tissues like brain and liver are quite softand require a gentler homogenization
approach in order to ensure adequate
sample extraction without sacrificing
the molecular integrity of the proteinsof interest. This is especially importantfor proteomic studies aiming to analyze
tertiary structure, enzyme activity or posttranslational modifications.

Equipment

The Bead Mill 24 is a bead mill homogenizer
capable of processing up to 24 tissue
samples per cycle and has been used in a
number of studies for the isolation ofproteins from soft tissues. 1,2,3 Bead mills
function by the high speed shaking of asample in a sealed tube in the presenceof beads. The beads impact the sample
with sufficient force to dissociate both thetissues and the cells.
In this study, we demonstrate protein
extraction from soft tissues through beadmilling using the Bead Mill 24. Efficiency
and analyte integrity were evaluated.

•Bead Mill 24 (Cat No. 15340163)•2 mL Soft Tissue Homogenizing Mix(Cat No. 15340153)

Sample size

Speed (m/s)

TimeBrain (70 mg)

4.85 m/s

20 secLiver (70 mg)

5.30 m/s

20 secLung (68 mg)

5.50 m/s

20 secSpleen (58 mg)

4.85 m/s

20 sec(mg)

Table 1 Sample size and Bead Mill 24 Settings

Procedure
Rattus norvegicus brain, liver, lung andspleen samples were placed into 2 mL nonreinforced tubes containing 0.57g of 1.4 mm
ceramic beads (Cat No. 15340153). 500 µL ofTris-buffered saline was added to each tube
and the samples were homogenized on theBead Mill 24 for one cycle at the settings
described in Table 1. Processing speed andduration were adjusted based on thetoughness and size of the tissue samples(Table 1). Homogenized mixtures wereimmediately centrifuged at 8,000 x g for 10minutes. 1.5 μl of the supernatant was used
to determine protein concentrations at 280
nm on a Nanodrop spectrophotometer(Thermo-Fisher).
2 μl of the protein extract was mixed with 5
μl Laemmli sample buffer(BioRad), heated at
95°C for 5 minutes, then 4 µL of each sample
and 5 µL of protein ladder were loaded andseparated by SDS-PAGE for 30 mins at 200V
on a 4-20% Tris Glycine SDS polyacrylamidegel (BioRad). Proteins were stained withCoomassie, destained and visualized on aGelDoc EZ system (BioRad).

Results
In this study, the Bead Mill 24’s ability toprocess a variety of soft tissue in order toextract proteins was evaluated. Highconcentrations of protein from all soft
tissue samples were detected post
homogenization. Concentrations weredetermined by spectrophotometry
as shown in Table 2. The highest
concentration was from spleen at
an average of 57.468 mg/mL and thelowest concentration was from brain at
10.687 mg/mL. SDS-PAGE analysis
indicating a broad protein repertoire wasobtained from each sample withabundant bands observed across theentire molecular weight range.

Sample Type
Brain

Concentration(mg/mL)10.687

Liver

24.438Lung

32.666

Spleen57.468

Table 2 Protein concentration of samples

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Lane 1: 10kD Ladder
Lane 2: Brain
Lane 3: Liver
Lane 4: Lung
Lane 5: Spleen

Figure 1: SDS-PAGE Analysis of soft tissue samples

ConclusionThe Bead Mill 24 is capable of homogenizing soft tissue samples in less than 30 seconds to obtain adequate protein yields as a starting
point for many proteomic applications. Due to the nature of different tissue types, different processing speeds and times were required
to complete sample disocation. The Bead Mill 24 was demonstrated to provide rapid protein extraction of up to 24 samples.

References
1. Shishido et al. “Bioavailability and Efficacy of a Gap Junction Enhancer (PQ7) in a Mouse Mammary Tumor Model.” Plos One 8.6 (2013).
Plos One. Web. 30 Sept. 2015. <http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067174>.
2. Bailey et al. “Inhibition of Hippocampal Aromatization Impairs Spatial Memory Performance in a Male Songbird.” Endocrinology 154.12(2013): 4707-14.
3. Chen et al. “Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitroand in Vivo.” Chemical Research in Toxicology 27.9 (2014): 1575-585.

Bead Mill 24: (Cat #15340163)