Trouble Shooting Zymogram - (Aug/02/2012 )

I am having problems getting my zymogram to work. I have no bands to speak of. I am making all solution fresh, with the exception of the 2xNRB. My sample comes from frozen heart tissue that has been infarcted.

Below is the protocol I am using.

Lysis Buffer: 1mM EDTA, 1mM EGTA, 10mM Tris, pH 7.5, 150mM NaCl, 1% Triton x-100, 0.5% Nonidet P-40, 0.2mM Sodium Orthovanadate,
2.5mM Sodium Pyrophosphate, 1mM Glycerol 2-phosphate Disodium Hyd
At the time of use:
Add 10 µl Halt Phosphatase Inhibitor Cocktail and
Add 4 µl PMSF (1mM) to 1ml Lysis Buffer.

Sample prep and Gel electrophoresis (`14 hours)
<*>Prepare 7.5 % Substrate separating gel with water that has 10 mg of gelatin per 4.0 mL.

<*>Add butanol on top of gel to get rid of the bubbles - Polymerize for 2 hours

<*>4% stacking Gel (~1 Hour).

<*>Load 2 ug (medium) or 50 ug (tissue) of protein. Add water to the necessary sample to give a constant volume for each sample. Final volume 30 uL (15 of which is 2xNRB)

<*>Add an equal volume of non-reducing buffer (do not boil)

10% SDS, 4% Sucrose, 0.1% Bromophenol Blue, 0.25 M Tris-HCl pH 6.85 ml stacking gel buffer qs to 10 ml.

<*>Run gel at 125 volts in 4° until the samples until the dye reaches the very bottom of the gel (~90 min).

<*>Shake gel with 2.5% triton (2.5mL/100mL) (with 0.02% NaN3 – 20 mg) 2 X for 30 min each to remove SDS and renature proteins.

Running buffer <192 mM glycine / 0.1% SDS / 25 mM Tris-HCl pH 8.3 >,
<*>Replace triton with dd water and shake for 2 x 10 min. Remove water and shake with in-gel enzyme buffer. This removes the triton. Rinse with a small amount of incubation buffer

<*>Place gel at 37⁰C in incubation buffer for 20 hours

Incubation buffer: 50 mM Tris-HCl pH 8.0, 5 mM CaCl2, 0.02% NaN3

Next Day
<*>Rinse for 5 mins with DI water.

<*>Take a picture of your gel while the markers are still visible.

<*>Replace with fresh commassie blue and shake for 30 min at room temp

<*>Replace with destain for 30 min (or longer) until you can see clear bands

40 % Methanol, 7% acetic acid

How visible are your marker bands? I would have thought that any protein bands would be fairly well diffused by this time, so I would have fixed the gel shortly after running.

bob1 on Thu Aug 2 21:19:05 2012 said:

Of course, that makes sense...

For the most part I have no bands. One of the lanes has a very faint band.

can you increase the sample?

I have tried loading 50 and 100 ug.

Could I be making a solution wrong? What would be the likely suspect?