APPLICATION NOTE #20145

Protein Extraction from Hard Tissue
Samples using the Bead Mill 24
Maliha Ishaq, Drexel Neumann, James A. Atwood PhD

The Bead Mill 24 enables rapid and efficienttissue homogenization for a variety ofsamples types. The Bead Mill 24 is capableof homogenizing up to twenty-four
samples in tube sizes ranging from 0.5 mL
to 50 mL using optimized bead matrices tofacilitate sample disruption. Hard samples
such as muscle, skin and bone represent asignificant challenge for extraction oftarget analytes such as proteins andnucleic acids.
Proteins are an important target inscientific research. They serve as
biomarkers for disease, targets fortherapeutic purposes, and provide
essential physiological functions in livingorganisms. Extraction and recovery of asample’s proteome is often the first stepin proteomic research. However, tissue
composition variability creates a significant
barrier for developing a universal
extraction method. Although soft tissue
types are easier to disrupt, hard tissues
such as bone, skin, and muscles are difficult
to disrupt quickly without degrading theanalyte of interest.
Traditional disruption methods can include
incubation in the presence of harshchemicals or pulverization under cryogenic
conditions. 1,2 For optimal protein recovery,
an efficient and reproducible method isnecessary that is effective across a broad
range of sample types.
Herein, we demonstrate protein extraction
from tough tissues via bead milling in theBead Mill 24.

Materials and Methods

Results

All rat tissues were obtained from
Bioreclamation, Inc . Skin, tail, tongue andback muscle samples were manuallysectioned and placed into 2 mL reinforced
polypropylene tubes filled with five 2.8
mm ceramic beads (Cat #15340154). 1 mLof Tris-HCl, pH 7.6 was added to each tube
and the samples were homogenized on
the Bead Mill as described in Table 1

Protein extraction from tough tissues suchas skin and muscle represent a significant
challenge and bottleneck for proteomic
research. Traditional approaches suchas chemical lysis or cyromilling are not
reproducible and are difficult to scale.
Bead milling, as supported in the Bead Mill24, increases protein extraction
throughput and enables homogenizationof tough biological tissues.

Homogenized mixtures were immediately
centrifuged at 8,000 g for 10 minutes. 1.2 µlof the supernatant was used to determine
protein concentrations at 280 nm on a
Nanodrop spectrophotometer (Table 1).
10 µl of the protein extract was mixed
with 5 µl Laemmli sample buffer, heatedat 90°C for 10 mins, then proteins wereseparated by electrophoresis for 30 mins
at 200V on a 4-20% Tris Glycine SDS
polyacrylamide gel. Proteins were stainedwith coomassie and visualized on a
GelDoc EZ system (BioRad).

Table 1 Sample Sizeand Bead Mill 24 Settings
Sample Type(mg)Speed(m/s)

CycleTime (s)Skin (90)

7.3

45 s

Tail (70)645 s

Tongue (90)7.45

3 x 30 s

Back Muscle (45)645 sBone (250)

6.95

8 x 45 s(45 s dwell)In this study rat skin, tail, tongue, muscle
and bone tissues were homogenized in
the Bead Mill 24 and protein yields werequantified by spectrophotometry. Table 2indicates that bead milling resulted inhigh protein yields from all tissue types
including bone.
Protein extracts were further separated
and visualized by SDS-PAGE (Figure 1).
Skin and tail produced very similar protein
repertoires due to the fact that both tissue
types are similar in that the tail is largely
composed of skin tissues. Tongue tissue,
while requiring the most amount of force
to generate a full homogenate, generated
a significant protein yield and the largest
molecular weight variation among its
visualized proteins (Table 2, Figure 1).
Protein extraction from bone samples
represent a unique challenge and are
typically achieved through a chemical
decalcification process. In this study fullhomogenization was achieved directly
through bead beating without the needthe tedious chemical lysis process. Bothhigh protein yields and a broad protein
repertoire was achieved through the beadmilling process (Table 2, Figure 2).

Table 2 Sample Type and Protein Yield
Sample TypeProtein Yield (µg/µL)

Skin

2.374

Tail

18.204

Tongue1.286

Back Muscle

14.395

Bone0.722

Fisher Scientific Bead Mill 24

Conclusion

Figure 2 Protein Gel Electrophoresis
on Tough Tissues. Polyacrylamide SDS
PAGE electrophoresis of protein extractions.
Lane 1: protein ladder. Lane 2: skin. Lane 3:tail. Lane 4: back muscle. Lane 5: tongue.

Figure 3 Protein Gel Electrophoresis
on Bone Protein Extracts. Polyacrylamide
SDS PAGE electrophoresis of bone protein
extractions. Lane 1: protein ladder.
Lane 2: bone protein extract.
Lane 3: 2X dilution of bone protein extract.
Lane 4: 3X dilution of bone protein extract.

The Bead Mill 24 facilitates rapid protein
extraction from tough tissues and bone.
Bead milling eliminates the need forstrong acids or expensive buffer systems
and provides for reproducible
homogenization.

References
1.

Jiang X et al. Method Developmentof Efficient Protein Extraction inBone Tissue for Proteome Analysis.
Journal of Proteome Research 2007 6:
2287-2294.

2.

Zakharchenko O et al. Optimized
Protocol for Protein Extraction fromthe Breast Tissue that is Compatible
with Two-Dimensional Gel
Electrophoresis. Breast Cancer: Basicand Clinical Research 2011 5: 37-42.

Part Numbers Referenced
Bead Mill 24 Motor Unit: 15340163
2 mL Pre-Filled Hard Tissue Homogenizing Mix:
15340157