buffer for ELISA

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nani
nani's picture
buffer for ELISA

Dear All
I need to make a lysis buffer to run ELISA on cell lysate.I have some difficulty in preparing it. I'll be appreciated if you could give me some hints esp about sodium orthovanadate.By the way, should I autoclave my buffer before adding aprotinin and leupeptin?
Here is the recipe:
1% NP-40, 20 mM Tris (pH 8.0), 137 mM NaCl , 10% glycerol, 2mM EDTA, 1mM activated sodium orthovanadate,10 mcg/mL Aprotinin,10 mcg/mL Leupeptin.
Cheers

Chin Fen Teo
Chin Fen Teo's picture
Hi Farnoush,

Hi Farnoush,

To prepare this kind of buffer, one could prepare stock buffers of each component and mix them to make their final concentrations accordingly. It depends on the nature of the component, some of them, I prefer to prepare them with autoclaved dd-water, some of them I just filter sterile.

I believe everyone has their own preference, but this is the list of stock that I would prepare:
10% NP40 (with autoclaved dd-water), 80% glycerol (with autoclaved dd-water), 0.5 M EDTA (autoclaved), 1 M Tris (sterile filter), 5 M NaCl (sterile filter), 0.1 M Na3VO4. And of course Aprotinin and Leupeptin.

If you want to prepare a master stock, I recommend to prepare one without Na3VO4, aprotinin and leupeptin, and supplement these components right before you need to use the buffer.

To prepare Na3VO4, you have to adjust the pH to 10 and boil the solution. I follow the instruction found on this page.

Good luck.