OHP assay Mod. 6/15/10 LMD Dourte from Ansorge, Erickson, Boxburger Hydroxyproline Assay for Collagens

Transcription

1 Hydroxyproline Assay for Collagens Day 1: Dry tubes 1.5mL microcentrifuge tubes (Fisher, Cat. # ) 1. Get 1.5mL microcentrifuge tubes, 1 for each sample, wear gloves when handling 2. Dry tubes for 24 hours at 65C, with the caps off (or 2 hrs lyophilizer) Day 2: Dry tube weight/wet sample weight Dissection instruments Kim Wipes 1. Tube Weights a. Remove tubes from oven, allow to cool for ~ 10mins (will effect the weight if not cooled) b. Weigh each tube to an accuracy of 5 decimal places c. Weigh each tube 3 times, be sure to re-zero the balance often (tare every 3) 2. Sample dissection a. Under dissection microscope, expose mouse patellar tendon, remove overlying tissue and separate sides from surrounding tissue. Divide longitudinally, use micro scissors to remove half of tendon from bone (treat other half of tendon as dictated by study design). Ensure all other non-tendon tissue has been removed under dissection scope. b. Place in petri dish with PBS. Blot sample on Kim wipe for ~3 seconds. c. Place in eppendorf tube and close lid 3. Wet weights a. After all dissections are complete, reweigh each tube 3 times 4. Freeze for storage a. Place in -80C until ready to complete assay 5. Dehydrate Samples a. Thaw samples. b. Uncap all tubes and place in lyophilizer (at least 2hrs) (or 65C for 48 hours) Day 3: Proteinase K digestion Proteinase K (see recipe at end) Ammonium Acetate (see recipe at end) 1. Dry weights a. Recap tubes and re-weigh each tube 3 times 2. Proteinase K Digestion a. Add 500 ul of 0.1M Ammonium Acetate to each sample b. Add 400 ul of 5mg/mL Proteinase K solution to each tube c. Finger flick each tube and give a brief spin on vortexer (excessive mixing is bad!)

2 d. Incubate samples at 65C for 16 hours (or can use water bath) Day 4: Acid Hydrolysis Proteinase K Ammonium Acetate 2mL Wheaton sealable glass ampoules 12N Hydrochloric acid (HCl) Repeater pipet and 5ml displacement tips Propane torch Metal tongs 1. Proteinase K Digestion a. Take samples out of oven b. Add an additional 100 ul of 5mg/ml proteinase K (gives total volume of 1mL which is important in analysis calculations) c. Incubate for an additional 1.5 hours d. Turn oven to 100 C and incubate for 10 minutes (to inactivate enzyme) e. Take tubes out and allow to cool 2. OHP alliquot a. Vortex samples b. Remove 100uL and place in labeled Wheaton ampoules i. If not using now place in microcentrifuge tube and store at -20C; Thaw at room temp when ready to continue ii. Remaining 900 ul is for proteoglycan analysis (or other test), store at -20C c. Add 500ul 12N HCl to each ampoule. Use the repeater pipet and do this step in the fume hood. (Ratio of 1:5, sample:hcl) d. Using the propane torch, heat the stem of the ampoule while gripping the end of the step with the tongs. Rotate the vial slowly in the jet for 10sec until the glass becomes malleable, then begin twisting the base of the ampoule. After a full twist, continue to twist and begin to pull the stem tip and base away from each other. Consistent and careful execution of this step will ensure the ampoules seal completely. e. Place the sealed ampoules on a heat block set at 110C for 16hrs or longer. Notes: - The hottest part of the flame is the tip of the jet. - After sealing the vials, place them in a white box with a divider. Then put a paper towel on the inside of the lid and close the box. Flip over for 10 seconds. Then check out the paper towel for wet spots. This trick will tell you if any of the vials aren t completely sealed. Day 5: Acid Neutralization and Drying Glass dessicator with cardboard divider Sodium hydroxide pellets (NaOH)

3 1. Remove ampoules and place in fume hood to cool for 30min (make sure they ve come to room temp before placing in vacuum so they don t boil!). 2. Using a wet paper towel, grip the stem and snap it off. 3. Place NaOH pellets in a weigh boat at the bottom of the dessicator. Transfer cooled, opened ampoules to the dessicator. 4. Attach dessicator to lyophilizer and turn on. Check to make sure oil level is adequate. Dry over NaOH until there is no moisture present (usually around 8hrs). 5. Rinse lyophilizer with DI water when finished Day 6: Sample Resuspension Repeat pipetter Parafilm Pasteur pipette and bulb 1. Remove dried samples from vacuum. Using the multishot, add 1ml of the assay buffer to each vial. (this will make it necessary to multiply by a dilution factor during analysis calculations) Seal with a strip of parafilm (be careful!) and vortex for 5s. 2. Store at 4C for 16h. Day 7: OHP Assay Orthohydroxyproline Assay Chloramine T # of plates *Chloramine T (grams) ddh20 (ml) n-propanol (ml) OHP Stock (ml) Volume (ml) DMAB # of plates **DMAB (grams) n-propanol (ml) Perchloric (ml) Volume (ml) *Chloramine T stored at 4C ** p-dimethylaminobenzaldehyde 1. Vortex again until any chunks have gone into solution. 2. Unwrap and transfer to prelabeled microcentrifuge tubes using a pasteur pipette and bulb. 3. Store samples at -20C or move on to OHP assay. 4. Set waterbath to 60C. Remove most of the water so that there s only about half a centimeter of water above the metal grating.

4 5. In a 50ml tube, dissolve chloramine T in ddh2o. Add propanol and the previously made stock buffer. Wrap tube with aluminum foil or store in a dark drawer. This solution needs to be made fresh the day of the assay. 6. In a small glass bottle, suspend DMAB in propanol. Slowly add perchloric acid. Wrap in aluminum foil or store in a dark drawer. This solution also needs to be made fresh the day of the assay. 7. Set up arrangement of plate prior to loading samples with standards on top in at least duplicate. Avoid using the outer rows and columns as they heat faster than the inner ones. 8. Uptake 150ul of a sample transfer to appropriate well. Do each sample in triplicate. 9. Put chloramine T in transfer well and use multi-channel pipetter to transfer 75ul of chloramine T reagent, mix by pipetting up and down to each well. Incubate at room temperature for 20min. Put a tube rack cover or another plate on top of your assay plate so that nothing drops into it. While waiting, turn on the microplate reader. 10. Add 75ul of the DMAB solution and mix by pipetting up and down. Place the carefully in the 60C waterbath for 7min. Transfer to a glass casserole dish partially filled with cold tap water and wait 5min. 11. Start the KC4 program and load the collagen assay protocol. Confirm that the plate layout matches your plate and that the read settings are set to 540nm absorbance. Remove the plate from the cold bath, dry the bottom of the plate with paper towels, and read it! 12. Export the data using the export function. Relabel the sample numbers with the correct plate layout. Notes: - Be sure to label the plates so that you don t mix them up. - When using the multichannel, be sure that the tips are pushed on all the way. - Set the waterbath to ~70C to maintain a 60C temperature with the lid open. Keep the lid open so that no water condenses and then drops into your plate. That would suck. Analysis: 1. Check raw data to ensure standard curve is appropriate 2. Create excel sheet with the following columns: Sample ID, 3 columns of tube weights, ave tube weight, 3 columns of wet weight, ave wet weight, Final wet weight (ave wet weight ave tube weight), 3 columns of dry weight, Final dry weight, corrected dry weight (ave dry weight ave tube weight), OHP conc (from output of plate reader), OH-P ave, OH-P st dev, Total Col (see below), Norm to wet weight (Tot col/final wet weight), Norm to dry weight (Tot col/final dry weight) 3. Set up the appropriate relationships/equations in the appropriate columns a. Total Col = Col ave*dilution ratio*7.143 i. Dilution ratio is inverse percent of original sample (eg. If after Prot K digestion, 100ul of 200ul was removed for HCl digestion, then the dilution ratio is 1/(100/200) = 2 ii comes from (Neuman and Logan 1950) 4. Insert data into appropriate locations to obtain final measurements of Col/wet weight and Col/dry weight a. Check for outliers in measurements, particularly in OH-P conc replicates

5 Solutions Needed: 0.1M Ammonium Acetate ml Ammonium Hydroxide (Normality = 14.8) ml Acetic Acid (Normality = 17.4) ~45 ml ddh2o Add all above ingredients. Bring ph to 7.0 with Ammonium hydroxide or acetic acid. Add dh2o to bring final volume to 50mL. Divide into ~5mL aliquots and freeze at -20C until needed. 5mg/mL Proteinase K Make enough for 1 use Add 5 mg of proteinase K (stored at -20C) per 1 ml of 0.1M Ammonium Acetate. Proteinase K sticks to everything so measure directly into tube. Freeze what is not used during the initial digestion. On second day of digestion, slowly thaw this solution in a cold water bath, then vortex only briefly and use. OHP Stock 25g Citric acid monohydrate 6ml Glacial acetic acid 60g Sodium acetate trihydrate 17g Sodium hydroxide pellets 500ml ddh2o Combine 25g citric acid monohydrate, 6ml acetic acid, 60g sodium acetate trihydrate, and 17g sodium hydroxide in 500mL of ddh2o. Adjust ph to 6.0. Store at 4C. OHP Buffer Dilute OHP Stock solution 1:10 in ddh2o. OHP Standards Hydroxyproline standard ddh20 OHP Buffer In a 50ml tube, dissolve 40mg of hydroxyproline in 40ml of ddh2o to make a 1mg/ml standard stock. Dilute this stock 1:10 by adding 1ml to 9ml assay buffer (1:10 dilution of stock buffer). Make the following standards: ug/ml by adding 2, 4, 6, etc ul of the 1:10 standard dilution to 998, 996, 994ul assay buffer (1:10 dilution of stock buffer). Make lower conc standards from higher (eg: take 100uL of 2mg/ml and add to 900uL OHP buffer for 0.2mg/mL standard). Be sure to vortex each vial or pipet up and down a lot. Neuman, R. E. and M. A. Logan (1950). "The determination of hydroxyproline." J Biol Chem 184(1):