papain is proteolytic enzyme. it digest protein. in your case u chondrocyte is linked to alginate by which bonding? for proteolysis, ug level is enough to degrade protein. I may have slipped my mind to what is

DMMB assay and sGAG. I hope i could help you more..

PAPAIN DIGESTION

PBE Buffer

100 mM Na2HPO4 (7.1g/500 mL DI-H2O)

10 mM EDTA (1.86g/500 mL DI-H2O)

pH 6.5

Papain Solution (in PBE buffer)

125 ug/ml papain

10 mM cysteine (0.035g/20mL)

50 U/mL hyaluronidase

Sample dehydration/digestion

1. Flash-freeze samples in liquid N2 for several minutes.

2. Put samples on lyophilizer until dry.

3. Measure dry weight of samples.

4. Prepare papain solution.

5. Add 1mL papain solution per sample (in microcentrifuge tube) - may have to vary amounts in have smaller/larger samples.

6. Cap tubes and incubate at 60C for 10-16 hours.

7. After incubation, vortex samples for several minutes.

8. Digested samples may be stored at –20C until ready to use.

DIMETHYLMETHYLENE BLUE (DMMB) ASSAY

-for the detection of sulfated glycosaminoglycans (GAGs)

DMMB solution: 16 ug DMMB/mL in glycine/NaCl solution (pH 3)

(store at room temp)

Glycine/NaCl solution: 3.04g glycine

2.37g NaCl

95 mL 0.1M HCl

905 mL DI-H2O

(makes 1000 mL total)

Chondroitin Sulfate (ChS) Standards:

-Dissolve 0.0875g cysteine in 50mL PBE, sterile filter

-To 1mL of PBE/Cys solution, add 50mg ChS

-Prepare working solution of 100 g/mL by diluting 0.1mL stock into 49.9mL PBE

(stable for several months at 4C)

-Prepare standards from working solution for 0, 1, 2, 3, 4, 5 g as follows:

g ChS L working soln L diH2O

0 0 50

1 10 40

2 20 30

3 30 20

4 40 10

5 50 0

*Linearity is only good up to 5g

PBE buffer: 100mM phosphate (7.1g Na2HPO4)

10mM EDTA (1.86g Na2EDTA)

500 mL DI-H2O

pH 6.5 (very important)

Procedure:

1. Set UV-Vis to =525 nm

2. Mix 50 L standards or sample+ 1.25 mL DMMB dye solution in a cuvette

3. Wipe outside of cuvette with a KimWipe and read absorbance immediately

Ref: Biochimica et Biophysica Acta 883 (1986) 173-177.

Thanks lei xu

Thanks to Lei Xu and Fadzilah. Actually, I am using 55mM sodium citrate, 5mM EDTA, 0.15M NaCl and 5mM cysteine HCl. and my papain concentration (orginal) is 100mg (sigma). I got information to use 0.56units/ml of papain. But I could not understand how to calculate that much papain from the 100mg original concentration. Should I calculate according to volume of dissolved bead (with dissolving buffer) ?

We calculate it according to the volume of the sodium citrate + bead solution. A 300ul sodium citrate sample requires approximately 3ul of 100x papain for a final concentration of 0.56U/ml.

Many many thanks to Ann Ouyang for your suggestion and kind help.

Hi Maruf, for the determination of our MSCs chondrogenic pellet GAG content we use an extratcion buffer made of Guanidine hydrochloride, sodiumacetate, 6-aminocapronicacid, EDTA, N-ethylmaleimid and protease inhibitor without an papain digestion. Instead we disrupt our pellet mechanically by a minilys precellys homogenizer. We worked quite a bit on improving our DMMB assay and that extraction method worked the best.

Alginate will also bind to DMMB (due to the negative charge of the alginate). You will have to adjust the pH of your DMMB solution in order to compensate for that (I think it's pH 1.5) so that the free H+ can competitively neutralize the charge on the alginate and the DMMB can then bind to the GAG molecules.

The relevant paper is:

Quantification of sulfated glycosaminoglycans in chondrocyte/alginate cultures, by use of 1,9-dimethylmethylene blue.

Enobakhare BO, Bader DL, Lee DA.

Anal Biochem. 1996 Dec 1;243(1):189-91.

http://www.ncbi.nlm.nih.gov/pubmed/8954546

Dear Maruf Minar

As per my experience pH of the solutions is more important in every steps. Find the pH of your solution before adding DMMB. if its more than 7 try to bring the pH to 6 range using PBE buffer. and do the dilutions of your serial standard with the same buffer.

if you look on this, definitly it ll help you to solve the problems.

This is my formula for the papain digest buffer which I use to beak down the alginate beads BEFORE the sample is tested in the DMMB assay

150 mM sodium chloride, 55 mM trisodium citrate, 5 mM EDTA (disodium salt), 5 mM L-cysteine hydrochloride and 0.56 Units/mL of papin enzyme. This buffer uses both papain to break down proteins in the sample, but also EDTA which will reverse the alginate gelling process (by binding calcium ions).

May I suggest the following article, which describes in detail the modifications to the typical DMMB assay required for alginate samples.

Enobakhare BO, Bader DL, Lee DA. Anal Biochem. 1996 Dec 1;243(1):189-9

Quantification of sulfated glycosaminoglycans in chondrocyte/alginate cultures, by use of 1,9-dimethylmethylene blue

Important points raised in this articule and to consider with alginate samples are

[1] use a lower pH of about 1.8 rather than usual pH 3 for the DMMB solution

[2] Ensure sufficient sample dilution to avoid saturating the DMMB solution with alginate

Best of luck with your experiment!