Erics Tasty Rat-Tail Collagen (Type I) Preparation

1. 2. 3. 4. 5. 6. Sterilize a 1 liter foil-covered beaker containing a Teflon magnetic stirbar. Place 5-10 tails in 95% ethanol to thaw. 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. When the tails have thawed, starting on the cut end of the tail, clamp 2 hemostats about 2-3 cm apart on a tail. While holding the hemostat in your left hand, twist or rotate the right hemostat 360 degrees and then pull. Keep pulling until it breaks off. You should have white collagen fibers at the end of the broken (2-3cm ) piece of tail. Cut the white fibers off the broken piece of the tail using a sharp razor blade and place them on glass gel plate. Continue breaking and pulling 2-3 cm pieces of the tail. You get less material the closer you get to the tip of the tail. Tease the collagen fibers by holding one end of the fibers stationary with one razor blade and then use a scraping motion with a razor blade at a 45 degree angle. You want to flatten the fibers and open them up. 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.

7. 8.

9.

10.

Stir in the cold room (maximum 2-3 days) until most of the collagen is dissolved and the viscosity of the solution is notably increased. Filter solution through sterile 70 m stainless steel mesh into sterile sample cups.

11.

12.

Put sufficient NaCl into a 1 liter Erlenmeyer to make a 3% solution when combined with the filtered collagen solution. Put a Teflon magnetic stirbar in the beaker. Autoclave.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Add filtered collagen solution to flask with NaCl and stir 1-2 h in the cold. Collect precipitate using sterile 250 ml bottles and the GSA rotor and centrifuging @ 6000 x g for 30 min at 4C. Discard supernatant Dissolve the pellets in a minimal amount of sterile 15 mM HCl (~75 ml total). solution will be very gooey. Add sterile NaCl to a final concentration of 3% (~ 9ml of a 30% solution). Transfer to Sarstedt 30 ml tubes (no need to sterilize) with caps and centrifuge at 10,000 x g for 30 min. Discard supernatant. 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. Centrifuge at 10,000 x g 10 min and discard ethanol. Drain well. Dissolve pellet in 20 mL of sterile 15 mM HCl. Centrifuge at 10,000 x g for 30 min and transfer supernatant to a new sterile 50 mL conical tube. Read 260/280 nm spectra. Use of 56,000 as the molar extinction coefficient for type I collagen ([(1)2(2)]. Ratio should be close to 1. Also check concentration by BCA assay. Dilute to ~ 4mg/ml with sterile 15mM HCl and put into sterile 50 ml tubes. Check sterility and polymerization.

Polymerization 1. 2. ON ICE! Dilute collagen to 0.5-1.0mg/ml in Hanks BSS, PBS, media, or sterile H2O. Add 1/10th volume of 10 X DMEM.

3.

Add up to 1/10th volume of collagen reconstitution buffer. Add until phenol red changes from yellow to light red or red-orange. If solution is still yellow add sterile 0.1 M NaOH until acid is neutralized. Solution can either be mixed with cells or put into chilled multiwell plates (setting on an ice pack in the hood) with cells added on top. Place in a 37C incubator 30-60 minutes to polymerize then overlay with medium. Embedding Adipocytes

4. 5.

1. 2. 3. 4. 5. 6.

Wash adipocytes 2-3 X in HBSS with 1%Fatty Acid Free BSA and 200 nM adenosine. 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. Add 1/10th volume of 10X DMEM to the collagen solution (1mg/ml). 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. Use the Eppendorf repeating pipettor to place 100 l of the collagen:adipocyte mix to each well. Put plate in 37 C incubator for 30-60 min to allow gelling to occur.

Plate Coating 1. 2. ON ICE! Dilute collagen to 0.1 mg/ml in HBSS. Add collagen reconstitution buffer until color changes to red/red-orange. 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. 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. Aspirate collagen solution. Wash each well with HBSS or PBS one or two times. Aspirate the wells and allow to dry 30-60 min in the hood with the lid off and the ultraviolet light on. Plates may be stored for long periods in the cold in a bag with a desiccant pack.

3. 4. 5. 6.

Recipes Collagen Reconstitution Buffer 2.2g sodium bicarbonate 4.8g HEPES 100mL H2O 15 mM HCl 1.2 ml concentrated HCl (12 M) H2O to 1 L

The Protein Sequence col1A1 rat

MFSFVDLRLL ICICHNGTAV EGPKGDPGPQ SQMSYGYDEK GGSGPMGPRG PGMKGHRGFS RPGPPGTAGA GSEGPQGVRG PGARGPSGPQ PAGEEGKRGA GAPGPAGPKG DGRPGPAGPP KDGEAGAQGA GVPGDLGAPG TGAPGAPGSQ ARGLTGPIGP GFAGPPGADG KGPRGAAGPP ETGPAGRPGE GLPGQRGERG SGREGSPGAE KNGDRGETGP GFSGLQGPPG IGPPGPRGRT GGRYYRADDA LKMCHSDWKS WYISPNPKEK ASQNITYHCK VDGCTSHTGT LLLGATALLT CDDVQCNEEL GPRGPVGPPG SAGVSVPGPM PPGPPGKNGD GLDGAKGDAG RGNDGAVGAA EPGPPGPAGA GPSGPPGPKG RGEPGPSGLP SPGEAGRPGE GARGQAGVMG PGPAGPAGER PSGARGERGF GAPGLQGMPG PGPAGAPGDK QPGAKGEPGD GATGFPGAAG VGPPGPPGPA FPGLPGPSGE GSPGRDGAPG AGPAGPIGPA SPGSPGEQGP GDSGPAGPPG NVVRDRDLEV GEYWIDPNQG KHVWFGESMT NSVAYMDQQT WGKTVIEYKT HGQEDIPEVS DCPNPQRREG RDGIPGQPGL GPSGPRGLPG DGEAGKPGRP PAGPKGEPGS GPPGPTGPTG AGPAGNPGAD NSGEPGAPGN GPPGERGGPG AGLPGAKGLT FPGPKGTAGE GEQGPAGSPG PGERGVQGPP ERGAAGLPGP GEAGPSGPPG TGVKGDAGPP RVGPPGPSGN GEKGSPGADG PGKQGPSGSS AKGDRGETGP GARGPAGPQG SGASGPAGPR PPGPPGPPGP DTTLKSLSQQ CNLDAIKVYC DGFPFEYGSE GNLKKALLLQ TKTSRLPIID CFV CIHNGLRVPN ECCAFCPEEY PGPPGPPGPP PPGAPGPQGF GERGPPGPQG PGENGAPGQM PPGFPGAVGA GQPGAKGANG KGDTGAKGEP SRGFPGADGV GSPGSPGPDG PGKAGERGLP FQGLPGPAGP GPAGPRGNNG KGDRGDAGPK PTGARGAPGD GPAGPAGPPG AGPPGPPGPV PAGSPGTPGP GERGPPGPMG AGPPGAPGAP PRGDKGETGE GPPGSAGSPG PSGGYDFSFL IENIRSPEGS NMETGQTCVF GSDPADVAIQ GSNEIELRGE VAPLDIGAPD GETWKPEVCL VSPNSEDVGV GPPGLGGNFA QGPPGEPGEP ARGLPGTAGL GPRGLPGERG KGEAGPQGAR APGIAGAPGF GATGVQGPPG AGPKGPSGER KTGPPGPAGQ GPPGAVGPAG PGEAGKPGEQ APGNDGAKGD GADGSPGKDG RGEAGPPGPA PIGNVGAPGP GKEGGKGPRG QGIAGQRGVV PPGLAGPPGE GAPGPVGPAG QGDRGIKGHR KDGLNGLPGP PQPPQEKSQD RKNPARTCRD PTQPSVPQKN LTFLRLMSTE GNSRFTYSTL QEFGLDIGPA

Amino Acid Glycine Alanine

number %age 389 130 26.77 8.95

Amino Acid Serine Threonine

number %age 67 44 4.61 3.03

Valine Leucine Isoleucine Phenylalanine Tyrosine Tryptophan Cysteine Methionine

42 51 25 26 14 6 18 14

2.89 3.51 1.72 1.79 0.96 0.41 1.24 0.96

Asparagine Glutamine Aspartic Acid Glutamic Acid Histidine Lysine Arginine Proline

34 48 60 76 9 55 69 276

2.34 3.30 4.13 5.23 0.62 3.79 4.75 19.00

The molecular weight using average isotopic mass is 138033.3 g/mol The total number of amino acids is The formal charge is The molar absorbance is 1453 -12 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 rat

MLSFVDTRTL GRDGVDGPVG LMGPRGPPGA GHPGKPGRPG QGVKGEPGAP PAGPIGSAGP GPPGNPGANG VGEPGPAGSK PPGLRGSPGS GLMGPRGLPG PGPKGPSGDP EQGPAGPPGF GESGAAGPSG RGAAGIPGGK EAGAAGPSGP GPKGENGIVG TGPPGPSGIT EKGPSGEPGT GPLGIAGPPG LLLAVTSCLA PPGPPGAPGP VGAPGPQGFQ ERGVVGPQGA GENGTPGQAG PGFPGAPGPK LTGAKGATGL GETGNKGEPG RGLPGADGRA SPGNVGPAGK GKPGEKGHPG QGLPGPSGTA PIGIRGPSGA GEKGETGLRG AGPRGSPGER PTGPVGAAGP GPPGPPGAAG TGPPGTAGPQ ARGPPGAVGS TCQSLQMGSV PGPPGPPGLT GPAGEPGEPG RGFPGTPGLP ARGLPGERGR GELGPVGNPG PGVAGAPGLP SAGAQGPPGP GVMGPPGNRG EGPVGLPGID LAGARGAPGP GEVGKPGERG PGPDGNKGEA EIGNPGRDGA GEVGPAGPNG SGPNGPPGPA KEGIRGPRGD GLLGAPGILG PGVNGAPGEA RKGPTGDRGP GNFAAQYSDK QTGPAGSRGP GFKGIRGHNG VGAPGPAGAR PAGPAGPRGE GPRGIPGPVG SGEEGKRGSP STGPAGVRGP GRPGPIGPAG DGNNGAQGPP LPGEFGLPGP GAVGAPGSAG RGAPGAIGAP FAGPAGSAGQ GSRGDGGPPG QGPVGRTGEI LPGSRGERGQ GRDGNPGSDG RGQRGPAGPR GVSAGPGPMG AGPPGKAGED LDGLKGQPGA GSDGSVGPVG AGLPGLSGPV AAGATGPRGL GEPGSAGPAG NGDAGRPGEP PRGEAGNIGF GPQGVQGGKG AGPRGERGPP ASGPGGLPGE GPAGASGDRG PGAKGEKGTK MTGFPGAAGR GASGPPGFAG PGIAGALGEP PPGRDGQPGH

KGERGYPGNI PRGPSGPQGI GPVGPAGPRG PGPPGPPGVS QIETLLTPEG CDFSTGETCI VSSKEMATQL SNDVELVAEG

GPTGAAGAPG PHGSVGPAGK HGNRGEPGPA RGDKGEPGDK GARGLPGLKG HNGLQGLPGL PAGPSGPIGK DGRSGHPGPV GPAGVRGSQG GGGYDFGFEG GFYRADQPRS QPSLRPKDYE SRKNPARTCR DLRLSHPEWK SDYYWIDPNQ QAQPVNTPAK NAYSRAQANK HVWLGETING AFMRLLANRA SQNITYHCKN SIAYLDEETG NSRFTYTVLV DGCSKKTNEW DKTVIEYKTN APLDIGGTNQ EFRVEVGPVC FK

GSVGPVGAVG AGLHGDQGAP SQGPAGPPGP VDATLKSLNN GCTMDAIKVY GSQFEYNAEG RLNKAVILQG KPSRLPFLDI

Amino Acid Glycine Alanine Valine Leucine Isoleucine Phenylalanine Tyrosine Tryptophan Cysteine Methionine

number %age 382 128 53 62 32 22 14 4 9 10 27.84 9.33 3.86 4.52 2.33 1.60 1.02 0.29 0.66 0.73

Amino Acid Serine Threonine Asparagine Glutamine Aspartic Acid Glutamic Acid Histidine Lysine Arginine Proline

number %age 64 44 41 39 40 63 12 49 74 230 4.66 3.21 2.99 2.84 2.92 4.59 0.87 3.57 5.39 16.76

The molecular weight using average isotopic mass is 129564.7 g/mol The total number of amino acids is The formal charge is The molar absorbance is 1372 +20 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