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Connective Tissue

Research Group
CTR CIA Protocols
CIA Protocols
Collagen Core
Animal Core
Molecular Resource Core
Rheumatic Disease Research Core
Center
Support Protocol 1: Purification of Type II
Collagen
Type II collagen (CII) is present only in hyaline cartilage and the vitreous humor
of the eye. When large quantities of CII are desired, fetal bovine or porcine
articular cartilages are convenient sources and can be purchased from
slaughterhouses. Sternal cartilage from young chickens is also a good source of
CII and can be obtained from wholesale meat markets that process chicken
breasts. In general, only negligible quantities of native CII can be solubilized from
normal cartilage using nondenaturing solvents.
This protocol describes the purification of CII from chick and fetal bovine cartilage.
CII can be purified from the cartilage of other species (e.g., rat, mouse, or pig;
Table 15.5.1) using the same procedure.
Table 15.5.1 Sources of Type II Collagen
Species Suggested tissue
Chicken Sternum
Bovine Knee joint
Porcine Knee joint
Rat Knee, hip, shoulder points
Xiphoid process and costal cartilage
Mouse Sternochondral cartilage
Regardless of the source, however, care must be taken to remove all sources of
type I collagen--e.g., the perichondrium--prior to extraction of the CII. Ideally,
100 to 400 g of cartilage should be processed at one time, given the time required
for the entire procedure. Smaller quantities can be processed (5 to 10 g) when the
quantity of cartilage is limited--e.g., when processing from rat or mouse sternums.
It is critical to minimize contamination of CII with type I collagen.
Materials
Fetal bovine joints or chick sternums
70% (v/v) isopropyl alcohol
Tris-buffered guanidine (see recipe)
10 mM, 100 mM, and 500 mM acetic acid
70% (v/v) formic acid
Pepsin (3x crystallized; Sigma)
5 M NaCl (APPENDIX 2)
10 mM Na2HPO4
Tris-buffered saline (TBS): 50 mM Tris_Cl, pH 7.4 (APPENDIX 2)/200 mM
NaCl
DE-52 anion-exchange resin (Whatman)
Scalpel or single-edged razor blade
Food processor or blender with sharp blades
Sorvall RC-5B or equivalent centrifuge with 250-ml centrifuge tubes and
1-liter centrifuge bottles
Dialysis tubing (MWCO 10,000)
5 x 20-cm chromatography column
5% SDS-PAGE gel (UNIT 8.4)
Additional reagents and equipment for dialysis (APPENDIX 3H), DE-52
ion-exchange chromatography (as for IgG purification; UNIT 2.7), and
SDS-PAGE (UNIT 8.4)
Contact Us
RDRCC
Arnold Postlethwaite, Director
Phone: 901-448-4979
Email: apostlet@uthsc.edu
Principal Investigators:
David Brand, PhD
Monica L. Brown, DO
Hongbo Chi, PhD
Weikuan Gu, PhD
Karen Hasty, PhD
Andrew H. Kang, MD
Linda K. Myers, MD
Eugene Pinkhassik, PhD
Arnold Postlethwaite, MD
Edward Rosloniec, PhD
Andrzej Slominski, MD, PhD
John Stuart, MD
Ae-Kyeung Yi, PhD
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CAUTION: Special precautions need to be taken when working with
fetal bovine and chicken tissue. Bovine tissue is a potential source of
infection with Brucella abortus, and chicken tissue may be
contaminated with Salmonella or Campylobacter. Gloves, mask and
eye protection should be worn and the tissue rinsed liberally with
70% isopropyl alcohol before dissection. Residual tissues should be
handled as potentially biohazardous material and disposed of
accordingly (see Chapter 7 introduction).
NOTE: All steps in this procedure must be performed at at 4C unless otherwise
indicated. Although it is nearly impossible to perform this procedure under totally
sterile conditions, maintaining the collagen preparation at such a low temperature
will not only help to maintain its native form, but will also help reduce bacterial or
mycobacterial growth.
Excise and Process cartilage
Rinse tissue liberally with 70% isopropyl alcohol to disinfect. Carefully dissect
the articular cartilage from bovine joints, meticulously freeing it from bone
and other noncartilaginous tissues.
A scalpel or a single-edged razor blade is recommended for gross
dissection and excision. The cartilage is identifiable as a firm,
elastic, translucent tissue that is easily sliced. Noncartilaginous
tissues are tough, white, nonelastic, and more difficult to cut.
Care should be taken to avoid collecting fibrocartilage, as it
contains potentially contaminating type I collagen.
When working with chicken sternums, removal of the
perichondrium (which is rich in potentially contaminating type I
collagen) can be facilitated by overnight incubation at 4C in 50
mM Tris_Cl (APPENDIX 2) containing 1 M NaCl. The remaining
cartilage is easily removed from the sternum by hand.
1.
Cut the cartilage into ~5-mm pieces using a scalpel. Using a food processor
or blender, mince the cartilage pieces as finely as possible.
This process is facilitated by the use of just enough cold distilled
water and chips of ice to cover the cartilage pieces. Ice chips are
required to keep the temperature of the cartilage pieces below
4C. Although the cartilage will not homogenize, the smaller the
size of the pieces obtained, the greater the yield of CII.
2.
Remove the water by centrifuging in 1-liter centrifuge bottles, 45 to 60 min
at 4600 x g, at 4C, using a Sorvall RC-5B or equivalent centrifuge. Stir the
minced cartilage overnight at 4C with 10 ml of Tris-buffered guanidine per
gram of minced cartilage.
This step removes much of the proteoglycan.
3.
Collect the minced cartilage by centrifuging in 1-liter centrifuge bottles, 30
min at 4600 x g, at 4C, then remove the supernatant. Wash three times to
remove the guanidine, each time by adding 10 to 15 ml cold distilled water
per gram of minced cartilage, centrifuging 30 min at 4600 x g, at 4C, then
discarding the supernatant.
Solubilize and purify CII.
4.
Resuspend the cartilage pellet in 20 vol of 500 mM acetic acid and adjust the
pH of the suspension to 2.8 using 70% formic acid.
5.
Add 1 g pepsin for every 20 g (wet weight) of cartilage and stir the
suspension gently for 36 to 48 hr in the cold.
The pepsin can be added as dry weight or first dissolved in cold
6.
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distilled water or 0.5 M acetic acid. The optimal pepsin:cartilage
ratio is 1:20 (weight/wet weight). The pepsin will digest the
cartilage and release soluble CII, causing the solution to become
viscous.
Separate the solubilized CII from the cartilage residue by centrifuging in
1-liter bottles, 30 min at 5000 x g, at 4C. Discard the pellet.
If separation is poor because of high viscosity, dilute with
additional 500 mM acetic acid and repeat centrifugation.
7.
Add 5 M NaCl to the supernatant slowly (over a 30 to 45 min period) to a
final concentration of 0.8 M. Allow collagen to precipitate at 4C for 12 to 24
hr.
8.
Recover the collagen precipitate by centrifuging in 1-liter bottles, 60 min at
4600 x g, 4C. Discard supernatant. Dissolve precipitate in 400 ml of 100 mM
acetic acid by stirring overnight at 4C.
9.
Centrifuge the collagen solution in 250-ml centrifuge bottles, 1 hr at 5000 x
g to remove any insoluble material.
If the solution is highly viscous, dilute with additional 100 mM
acetic acid before centrifugation.
10.
Repeat the salt precipitation of the CII (steps 8 to 10) three additional times
and dissolve the last collagen precipitate in 400 ml of 100 mM acetic acid.
These steps are necessary to obtain highly purified CII. The
volume of acetic acid depends on the initial amount of cartilage,
assumed to be 200 g in this protocol.
11.
Precipitate the collagen by dialysis against several changes of 10 mM
Na2HPO4 at 4C using MWCO 10,000 dialysis tubing (see APPENDIX 3H).
Collect the collagen precipitate by centrifuging in 250-ml bottles, 40 min at
9000 x g, 4C, then wash the precipitate twice each time by centrifuging 40
min at 9000 x g, removing the supernatant, adding 200 ml of 10 mM
Na2HPO4 to the pellet, then centrifuging again and removing the
supernatant.
This step inactivates and helps remove any residual pepsin.
12.
Solubilize the collagen precipitate in 400 ml TBS, then dialyze overnight
against TBS at 4C using MWCO 10,000 dialysis tubing (APPENDIX 3H).
13.
Pass the dialyzed solution over a preswollen 5 x 20-cm DE-52 column which
has been equilibrated with TBS (see UNIT 2.7; use TBS in place of Tris_Cl).
CII elutes from the DE-52 column unretarded; residual
proteoglycans and pepsin are retained by the resin.
14.
Precipitate the collagen from the column effluent by adding 5 M NaCl slowly
(over a 30- to 45-min period) to a final concentration of 2.5 M. Allow collagen
to precipitate at 4C for 12 to 24 hr.
15.
Collect the collagen precipitate by centrifugation in 250-ml centrifuge bottles
for 60 min at 4000 x g, 4C and remove the supernatant. Dissolve the pellet
by stirring in 400 ml of 100 mM acetic acid, then dialyze exhaustively at 4C
against 10 mM acetic acid using MWCO 10,000 dialysis tubing to remove the
salt. Lyophilize desalted CII and store at -20C (stable _3 years if handled
properly).
The volume of acetic acid used at this step depends on the initial
amount of cartilage; see step 11, annotation.
See Critical Parameters for additional guidelines on how to store
the CII.
16.
Determine purity of CII by SDS-PAGE using a 5% gel (see UNIT 8.4). 17.
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