uthsc _ ctr _ collagen-induced arthritis (cia) _ support protocol 1

7/27/2019 UTHSC _ CTR _ Collagen-Induced Arthritis (CIA) _ Support Protocol 1

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nnective Tissuesearch Group

CTR CIA Protocols

A Protocols

ollagen Core

nimal Core

olecular Resource Core

eumatic Disease Research Corenter

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, Dir

Phone: 901-448-4979

Email: apostlet@uthsc.

Principal Investigato

David Brand, PhD

Monica L. Brown, DO

Hongbo Chi, PhD

Weikuan Gu, PhD

Karen Hasty, PhD

Andrew H. Kang, MD

Linda K. Myers, MDEugene Pinkhassik, PhD

Arnold Postlethwaite, MD

Edward Rosloniec, PhD

Andrzej Slominski, MD, P

John Stuart, MD

Ae-Kyeung Yi, PhD

https://www.uthsc.edu/ctr/cia_support_prot


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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 otherwiseindicated. 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.


uthsc _ ctr _ collagen-induced arthritis (cia) _ support protocol 1

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