collagen methods

8/6/2019 Collagen Methods

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Erics Tasty Rat-Tail Collagen (Type I) Preparation

1. Sterilize a 1 liter foil-covered beaker containing a Teflon magnetic stirbar.2. Place 5-10 tails in 95% ethanol to thaw.3. Prepare sterile 15 mM HCl by adding 1.2 ml of conc. HCl to 1 1iter of NanoPure

H2O. Filter through a 0.22 m filter into a sterile bottle.

4. When the tails have thawed, starting on the cut end of the tail, clamp 2 hemostatsabout 2-3 cm apart on a tail.

5. While holding the hemostat in your left hand, twist or rotate the right hemostat360 degrees and then pull. Keep pulling until it breaks off.

6. You should have white collagen fibers at the end of the broken (2-3cm ) piece oftail. Cut the white fibers off the broken piece of the tail using a sharp razor bladeand place them on glass gel plate.

7. Continue breaking and pulling 2-3 cm pieces of the tail. You get less material thecloser you get to the tip of the tail.

8. Tease the collagen fibers by holding one end of the fibers stationary with onerazor blade and then use a scraping motion with a razor blade at a 45 degreeangle. You want to flatten the fibers and open them up.

9. Put the teased fibers in the stirring 15 mM HCl. They should turn transluscent.When finished, put the beaker of collagen at 4oC and stir overnight or longer if

needed.

10. Stir in the cold room (maximum 2-3 days) until most of the collagen is dissolvedand the viscosity of the solution is notably increased.

11. Filter solution through sterile 70 m stainless steel mesh into sterile sample cups.


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12. Put sufficient NaCl into a 1 liter Erlenmeyer to make a 3% solution whencombined with the filtered collagen solution. Put a Teflon magnetic stirbar in the

beaker. Autoclave.

13.

Add filtered collagen solution to flask with NaCl and stir 1-2 h in the cold.

14. Collect precipitate using sterile 250 ml bottles and the GSA rotor and centrifuging@ 6000 x g for 30 min at 4C.

15. Discard supernatant16. Dissolve the pellets in a minimal amount of sterile 15 mM HCl (~75 ml total).

solution will be very gooey.

17. Add sterile NaCl to a final concentration of 3% (~ 9ml of a 30% solution).18. Transfer to Sarstedt 30 ml tubes (no need to sterilize) with caps and centrifuge at

10,000 x g for 30 min.

19. Discard supernatant.20. Disperse pellets into 20 mL of 70% ethanol per tube. Seal with cap and Parafilm

and put on the rocking platform in the cold 1-3 days.

21. Centrifuge at 10,000 x g 10 min and discard ethanol. Drain well.22. Dissolve pellet in 20 mL of sterile 15 mM HCl.23. Centrifuge at 10,000 x g for 30 min and transfer supernatant to a new sterile 50

mL conical tube.

24. Read 260/280 nm spectra. Use of 56,000 as the molar extinction coefficient fortype I collagen ([(1)2(2)]. Ratio should be close to 1.

25. Also check concentration by BCA assay.26. Dilute to ~ 4mg/ml with sterile 15mM HCl and put into sterile 50 ml tubes.27. Check sterility and polymerization.

Polymerization

1. ON ICE! Dilute collagen to 0.5-1.0mg/ml in Hanks BSS, PBS, media, or sterileH2O.

2. Add 1/10th volume of 10 X DMEM.


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3. Add up to 1/10th volume of collagen reconstitution buffer. Add until phenol redchanges from yellow to light red or red-orange. If solution is still yellow add

sterile 0.1 M NaOH until acid is neutralized.

4. Solution can either be mixed with cells or put into chilled multiwell plates (settingon an ice pack in the hood) with cells added on top.

5. Place in a 37C incubator 30-60 minutes to polymerize then overlay withmedium.

Embedding Adipocytes

1. Wash adipocytes 2-3 X in HBSS with 1%Fatty Acid Free BSA and 200 nMadenosine.

2. Put 1/10th volume of reconstitution buffer in each well of a multiwell plate. E.g.10 l into a 48 well plate well to which you will add 100 l of sample.

3. Add 1/10th volume of 10X DMEM to the collagen solution (1mg/ml).4. Add an equal volume of packed adipocytes to the collagen. Generally 3ml of

cells and 3 ml of collagen are enough for one 48 well plate.

5. Use the Eppendorf repeating pipettor to place 100 l of the collagen:adipocytemix to each well.

6. Put plate in 37 C incubator for 30-60 min to allow gelling to occur.

Plate Coating

1. ON ICE! Dilute collagen to 0.1 mg/ml in HBSS. Add collagen reconstitutionbuffer until color changes to red/red-orange.

2. Pipet into multiwell plates (500 uL for a six well, 100 uL for a 24 well, 50 uL/well for a 48 well, and 25 uL for a 96 well). Shake plate to make sure solution

completely covers the well bottom.

3. Wrap plate in original bag or Saran wrap and place in cold room overnight.Alternately plates can be incubated at ambient temp for 1-2 h.

4. Aspirate collagen solution. Wash each well with HBSS or PBS one or two times.5. Aspirate the wells and allow to dry 30-60 min in the hood with the lid off and the

ultraviolet light on.

6. Plates may be stored for long periods in the cold in a bag with a desiccant pack.


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Recipes

Collagen Reconstitution Buffer2.2g sodium bicarbonate4.8g HEPES

100mL H2O

15 mM HCl

1.2 ml concentrated HCl (12 M)

H2O to 1 L

The Protein Sequence col1A1 ratMFSFVDLRLL LLLGATALLT HGQEDIPEVS CIHNGLRVPN GETWKPEVCL

ICICHNGTAV CDDVQCNEEL DCPNPQRREG ECCAFCPEEY VSPNSEDVGV

EGPKGDPGPQ GPRGPVGPPG RDGIPGQPGL PGPPGPPGPP GPPGLGGNFA

SQMSYGYDEK SAGVSVPGPM GPSGPRGLPG PPGAPGPQGF QGPPGEPGEP

GGSGPMGPRG PPGPPGKNGD DGEAGKPGRP GERGPPGPQG ARGLPGTAGL

PGMKGHRGFS GLDGAKGDAG PAGPKGEPGS PGENGAPGQM GPRGLPGERG

RPGPPGTAGA RGNDGAVGAA GPPGPTGPTG PPGFPGAVGA KGEAGPQGAR

GSEGPQGVRG EPGPPGPAGA AGPAGNPGAD GQPGAKGANG APGIAGAPGF

PGARGPSGPQ GPSGPPGPKG NSGEPGAPGN KGDTGAKGEP GATGVQGPPG

PAGEEGKRGA RGEPGPSGLP GPPGERGGPG SRGFPGADGV AGPKGPSGER

GAPGPAGPKG SPGEAGRPGE AGLPGAKGLT GSPGSPGPDG KTGPPGPAGQ

DGRPGPAGPP GARGQAGVMG FPGPKGTAGE PGKAGERGLP GPPGAVGPAG

KDGEAGAQGA PGPAGPAGER GEQGPAGSPG FQGLPGPAGP PGEAGKPGEQ

GVPGDLGAPG PSGARGERGF PGERGVQGPP GPAGPRGNNG APGNDGAKGD

TGAPGAPGSQ GAPGLQGMPG ERGAAGLPGP KGDRGDAGPK GADGSPGKDGARGLTGPIGP PGPAGAPGDK GEAGPSGPPG PTGARGAPGD RGEAGPPGPA

GFAGPPGADG QPGAKGEPGD TGVKGDAGPP GPAGPAGPPG PIGNVGAPGP

KGPRGAAGPP GATGFPGAAG RVGPPGPSGN AGPPGPPGPV GKEGGKGPRG

ETGPAGRPGE VGPPGPPGPA GEKGSPGADG PAGSPGTPGP QGIAGQRGVV

GLPGQRGERG FPGLPGPSGE PGKQGPSGSS GERGPPGPMG PPGLAGPPGE

SGREGSPGAE GSPGRDGAPG AKGDRGETGP AGPPGAPGAP GAPGPVGPAG

KNGDRGETGP AGPAGPIGPA GARGPAGPQG PRGDKGETGE QGDRGIKGHR

GFSGLQGPPG SPGSPGEQGP SGASGPAGPR GPPGSAGSPG KDGLNGLPGP

IGPPGPRGRT GDSGPAGPPG PPGPPGPPGP PSGGYDFSFL PQPPQEKSQD

GGRYYRADDA NVVRDRDLEV DTTLKSLSQQ IENIRSPEGS RKNPARTCRD

LKMCHSDWKS GEYWIDPNQG CNLDAIKVYC NMETGQTCVF PTQPSVPQKN

WYISPNPKEK KHVWFGESMT DGFPFEYGSE GSDPADVAIQ LTFLRLMSTE

ASQNITYHCK NSVAYMDQQT GNLKKALLLQ GSNEIELRGE GNSRFTYSTL

VDGCTSHTGT WGKTVIEYKT TKTSRLPIID VAPLDIGAPD QEFGLDIGPA

CFV

Amino Acid number %age Amino Acid number %age

Glycine 389 26.77 Serine 67 4.61

Alanine 130 8.95 Threonine 44 3.03


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Valine 42 2.89 Asparagine 34 2.34

Leucine 51 3.51 Glutamine 48 3.30

Isoleucine 25 1.72 Aspartic Acid 60 4.13

Phenylalanine 26 1.79 Glutamic Acid 76 5.23

Tyrosine 14 0.96 Histidine 9 0.62

Tryptophan 6 0.41 Lysine 55 3.79

Cysteine 18 1.24 Arginine 69 4.75

Methionine 14 0.96 Proline 276 19.00

The molecular weight using average isotopic mass is 138033.3 g/mol

The total number of amino acids is 1453The formal charge is -12

The molar absorbance is 60402 (M*cm)-1

For an A280 of 1 in a 1cm cell and a 1-fold dilution

The stock solutions are

0.017 mM 2.29 mg/mL

The Protein Sequence col1A2 ratMLSFVDTRTL LLLAVTSCLA TCQSLQMGSV RKGPTGDRGP RGQRGPAGPR

GRDGVDGPVG PPGPPGAPGP PGPPGPPGLT GNFAAQYSDK GVSAGPGPMG

LMGPRGPPGA VGAPGPQGFQ GPAGEPGEPG QTGPAGSRGP AGPPGKAGED

GHPGKPGRPG ERGVVGPQGA RGFPGTPGLP GFKGIRGHNG LDGLKGQPGA

QGVKGEPGAP GENGTPGQAG ARGLPGERGR VGAPGPAGAR GSDGSVGPVG

PAGPIGSAGP PGFPGAPGPK GELGPVGNPG PAGPAGPRGE AGLPGLSGPV

GPPGNPGANG LTGAKGATGL PGVAGAPGLP GPRGIPGPVG AAGATGPRGL

VGEPGPAGSK GETGNKGEPG SAGAQGPPGP SGEEGKRGSP GEPGSAGPAG

PPGLRGSPGS RGLPGADGRA GVMGPPGNRG STGPAGVRGP NGDAGRPGEP

GLMGPRGLPG SPGNVGPAGK EGPVGLPGID GRPGPIGPAG PRGEAGNIGF

PGPKGPSGDP GKPGEKGHPG LAGARGAPGP DGNNGAQGPP GPQGVQGGKG

EQGPAGPPGF QGLPGPSGTA GEVGKPGERG LPGEFGLPGP AGPRGERGPP

GESGAAGPSG PIGIRGPSGA PGPDGNKGEA GAVGAPGSAG ASGPGGLPGE

RGAAGIPGGK GEKGETGLRG EIGNPGRDGA RGAPGAIGAP GPAGASGDRG

EAGAAGPSGP AGPRGSPGER GEVGPAGPNG FAGPAGSAGQ PGAKGEKGTK

GPKGENGIVG PTGPVGAAGP SGPNGPPGPA GSRGDGGPPG MTGFPGAAGR

TGPPGPSGIT GPPGPPGAAG KEGIRGPRGD QGPVGRTGEI GASGPPGFAG

EKGPSGEPGT TGPPGTAGPQ GLLGAPGILG LPGSRGERGQ PGIAGALGEP

GPLGIAGPPG ARGPPGAVGS PGVNGAPGEA GRDGNPGSDG PPGRDGQPGH


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KGERGYPGNI GPTGAAGAPG PHGSVGPAGK HGNRGEPGPA GSVGPVGAVG

PRGPSGPQGI RGDKGEPGDK GARGLPGLKG HNGLQGLPGL AGLHGDQGAP

GPVGPAGPRG PAGPSGPIGK DGRSGHPGPV GPAGVRGSQG SQGPAGPPGP

PGPPGPPGVS GGGYDFGFEG GFYRADQPRS QPSLRPKDYE VDATLKSLNN

QIETLLTPEG SRKNPARTCR DLRLSHPEWK SDYYWIDPNQ GCTMDAIKVY

CDFSTGETCI QAQPVNTPAK NAYSRAQANK HVWLGETING GSQFEYNAEG

VSSKEMATQL AFMRLLANRA SQNITYHCKN SIAYLDEETG RLNKAVILQG

SNDVELVAEG NSRFTYTVLV DGCSKKTNEW DKTVIEYKTN KPSRLPFLDI

APLDIGGTNQ EFRVEVGPVC FK

Amino Acid number %age Amino Acid number %age

Glycine 382 27.84 Serine 64 4.66

Alanine 128 9.33 Threonine 44 3.21

Valine 53 3.86 Asparagine 41 2.99

Leucine 62 4.52 Glutamine 39 2.84

Isoleucine 32 2.33 Aspartic Acid 40 2.92

Phenylalanine 22 1.60 Glutamic Acid 63 4.59

Tyrosine 14 1.02 Histidine 12 0.87

Tryptophan 4 0.29 Lysine 49 3.57

Cysteine 9 0.66 Arginine 74 5.39

Methionine 10 0.73 Proline 230 16.76

The molecular weight using average isotopic mass is 129564.7 g/molThe total number of amino acids is 1372

The formal charge is +20

The molar absorbance is 47414 (M*cm)-1

For an A280 of 1 in a 1cm cell and a 1-fold dilution

The stock solutions are

0.021 mM 2.73 mg/mL

collagen methods

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