REAGENT PREPARATION:
1X TTBS
Add 6.05 g Tris base (50 mM), 8.76 g sodium chloride (150 mM) to 800 mL of distilled water. Adjust
pH to 7.5 with HCl. Adjust to 1 liter with distilled water. Add Tween-20 to 0.1% (v/v). Maybe stored
for up to 3 months at room temperature.
Material Required but Not Supplied:
Solutions Required But Not Supplied
To make 1.0 Liter of TTBS:
800 mL of deionized water
Add 12.1 g of Tris base
Add 8.8 g of NaCl
Dissolve and adjust pH to 7.5 with HCl
Add 1.0 mL of Tween-20 (provided)
Bring volume up to 1.0 Liter with deionized water
Filter with 3 mM Whatmann paper or equivalent
Store at room temperature for no more than one month
To make 100 mL of TTBS with 1.0% (w/v) BSA:
100 mL TTBS
Add 1.0 g of BSA (provided)
Dissolve and use immediately
Gelatin Zymography
Gelatinase substrate gel electrophoresis was performed with the use of precast gels (10%
polyacrylamide containing 0.1% gelatin, Novex).
Samples were prepared by dilution (1:200) into a loading buffer consisting of 0.4 mol/L
Tris, pH 6.8, 5% SDS, 20% glycerol, 0.03% bromophenol blue, and 20 µL of serum
loaded per lane.
After electrophoresis at 125 V, the gels were incubated in renaturing solution (2.5%
Triton-X-100) for 30 minutes at room temperature and then for 72 hours at 37°C in a
developing buffer containing 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 4 mmol/L
CaCl2, and 0.02% Brij-35. When appropriate, the developing buffer contained 20 mmol/L
EDTA, a known inhibitor of MMPs, or l mmol/L PMSF, a known inhibitor of serine
proteases. The gels were then stained with Coomassie blue, and regions without staining
were indicative of gelatin lysis.
Serum samples were prepared as follows for activation with APMA. First 25 µL of 5
mmol/L APMA was added to 100 µL of undiluted serum and incubated in a 37°C water
bath for 90 minutes. The reaction was stopped with 10 mmol/L EDTA, and the samples
were dialyzed for 18 hours to remove the APMA. The samples were then loaded on gels,
and the zymography protocol was followed as above.
Zymography Protocol:
By:Dr. Ken Swanson
Zymography Protocol:
Zymography allows the detection and rapid identification of matrix metalloproteinase
(MMP) activities in biological samples. Samples containing MMP, including tumor and
cell lysates, and body fluids such as cerebral spinal fluid (CSF) are subjected to
separation by SDS-PAGE in a resolving gel containing a co-polymerized protease
substrate such as gelatin. After being resolved in this gel, proteases are re-natured and act
to hydrolyze the protein substrate in the gel matrix. The presence and relative activity of
MMP’s in the sample are thus determined by a decrease in Coomassie brilliant blue
staining of the digested gelatin at the position of the MMP. Since Coomassie® Brilliant
Blue fluoresces in the near infrared and can readily be detected within the 700 channel of
the LI-COR Odyssey, this MMP-dependent decrease in Coomassie staining can be
readily detected and quantified using the protocol below. This protocol allows for a more
precise determination of the relative amount of MMP activity than has previously been
possible by zymography and provides a more convenient means of acquiring, storing and
presenting this data.
Reagents:
Gelatin (Sigma-G8150)
Calcium Chloride(Sigma)
Tris-Acetic? Acid
Tris-Base?
Glycine
Triton X®-100
30% Acrylamide 0.8% Bis-Acrylamide?
TEMED
Ammonium Persulfate
Sodium Dodecylsulfate
Coomassie Brilliant Blue R-250 (Biorad 161-0406)
CSF zymography protocol
• 1.The resolving gel is made as follows: 40 mg of gelatin in 10 ml of deionized water
(70oC). In order, add 10 ml of deionized water (21oC) to cool the mixture, 10 ml of 4X
separating buffer (1.5M Tris HCl, pH 8.8) and 10 ml of 30% acrylamide 0.8% bisacrylamide (Sigma) solution. Initiate polymerization by the addition of 200 l of TEMED
(Sigma) and cast as al of ammonium persulfate (Sigma) and 50  mini-gel (8 cm X 5
cm X 0.75mm).
• 2. After the resolving gel is fully polymerized, pour the stacking gel (9 ml of distilled
water, 3.75 ml of stacking buffer (0.5M Tris HCl, pH 6.8), 2.25 ml of 30% acrylamide,
0.8% bis-acrylamide, l TEMED) and place the comb.l APS and the addition of 30200
• Denature samples containing the MMPs to be analyzed in 1X Laemmli sample buffer
with BME and electrophorese within the gel described above at 25 mA until desired
resolution is attained. Running buffer: 25 mM Tris pH 6.8, 190mM glycine and 3.5 mM
SDS in 500 ml of distilled water.
• After electrophoresis is complete, incubate the gel in 2.5% Trition X®-100 (Sigma)
washing buffer with shaking at 10 rpm for 1 hour.
• Incubate in digestion buffer (10 mM calcium chloride, 20mM Tris Acetic Acid with pH
of 7.5.) for 8 to 12 hours 37°C.
• Stain the gel for an hour in 26.6ml of 3X Coomassie Brilliant Blue R-250 (Biorad)
solution, (0.1% mass/vol of Coomassie Blue), with 13% acetic acid, De-stain in 10%
acetic acid and 5% methanol for 3 hours.
• Scan the de-stained gels on the LI-COR Odyssey® using the 700 channel. Scan at
intensity 5 as a starting point
• Quantify digested areas using the ‘Average’ background method in the Background
settinngs. Since the Odyssey is detecting a net decrease in intensity, maintain the same
size for each quantification shape that is drawn.
Notes:
• Optimal mass of material to be analyzed and time of digestion for each experimental
paradigm must be determined empirically. Bands that are completely digested in the
center diverge from linearity, thus over digestion should be avoided. It is suggested that a
concentration curve be generated at the beginning of a study using the Odyssey to
determine the optimal mass of the material being studied. Once established, the time and
temperature of the reactions must be adhered to during subsequent experiments.
• Incompletely dissolved gelatin (step 1) and low concentration of Coomassie Brilliant
Blue (step 5) may lead to uneven staining and poor quantification.
References
1. Perides G, Asher RA, Lark MW, et al. Glial hyaluronate-binding protein: a product of
metalloprotease digestion of versican? Biochem J 1995; 312: 377-384.
2. Perides G, Charness ME, Tanner LM, et al. Matrix metalloproteases in the
cerebrospinal fluid of patients with Lyme borreliosis. J Infect Dis 1998; 177: 401-408.
3. Friedberg MH, Glantz MJ, Klempner MS, et al. Specific matrix metalloprotease
profiles in the cerebrospinal fluid correlates with the presence of malignant astrocytomas,
brain metastases, and carcinomatous meningitis. Cancer 1998; 82: 923-930.
4. Timothy E, Van Meter TE, William C, et al. Induction of membrane-type-1 matrix
metalloproteinase by epidermal growth factor-mediated signaling in gliomas. NeuroOncol? 2004; 6(3):188-199
Coomassie Staining
0.2% Coomassie Gel Stain (in 7.5% acetic acid and 40% methanol):
0.2 g Coomassie
7.5 ml acetic acid
40 ml methanol
Q.S. with H2O to 100 ml. Nalgene or Whatman filter before use.
Coomassie Rapid Destain (7.5% acetic acid and 5% methanol):
75 ml acetic Acid
50 ml methanol
Q.S. with H20 to 1 L.