I was hoping to improve upon a cocktail that I currently use; RIPA lysis buffer (1x Modified RIPA lysis buffer: 10mM Tris-HCl, pH7.5; 10mM EDTA; 1% NP-40; 0.1% SDS; 150nM NaCl; 0.5% sodium deoxychloride) and to this I add 1% protease inhibitor, 1% sodium orthovanadate and 1 % NaF.
I am particularly interested to know the correct amount of NaF to use.
My limited experience. There are many different recipies for RIPA and I see no difference among them. I put phosphotase inhibitor cocktails 2 and 3 in my RIPA then use 1% BSA in TBS-T to block. Previously I used 5% skim milk in TBST but was having difficulty in detecting phospho-ERK but now is not an issue. I can even use MOPS buffer to get similar results. Hope my limited experience can help you.
I agree with Eva-Maria above that RIPA may not be the best buffer to use in this case. Here is my recipe for NP-40 lysis buffer and the concentrations of protease and phosphatase inhibitors that I make up individualyl and add just before use to the NP-40 buffer. Concentrations given are final concentrations:
NP-40 lysis buffer
Tris-HCl pH 7.5 50 mM
NaCl 150 mM
Nonident P-40 (NP-40) 1% v/v
EDTA 5 mM
Sodium Vanadate 1 mM
Sodium Molybdate 1 mM
Sodium Fluoride 10 mM
PMSF 40 μg/ml
Pepstatin A 0.7 μg/ml
Aprotinin 10 μg/ml
Leupeptin 10 μg/ml
Soybean Trypsin Inhibitor 10 μg/ml
Hi Salman,
In my Phospho-experiment, I'm using the following buffer:
Tris-HCl 20 mM pH7.5,
NaCl 150 mM,
EDTA 1 mM,
EGTA 1 mM,
Triton X-100 1%,
Sodium Pyrophosphate 2.5 mM,
beta-Glycerophosphate 1 mM,
NaF 30 mM.
Before use, I add freshly Na3VO4 1 mM, protease and phosphatase inhibitor cocktails.
For 1.5 millions cells, I add 250-300 µL of lysis buffer to the cell pellet, incubate 30 min at 4ºC while rotating. Clear the lysate at 14,000 rpm, 15 min, 4ºC.
For the WB,
I load 50 µg of whole cell lysate to the gel
I block the PVDF membrane in 5% BSA in TBS + Sodium azide 0.01 % + phenol red for 1-2 h, at RT while agitating.
The primary Ab is diluted in 5% BSA in TBS + Sodium azide 0.01 % + phenol red overnight, 4ºC while agitating.
Wash with TBS 6 x 5 min, RT, agitating.
Secondary Ab is diluted in 10 % milk + TBS.
I use primary Ab from Cell Signaling Technologies (product 4370), they are very clean, then no need of TBS-T. You can reuse the primary antibody solution in this setup at least 3 times !
Then I'm sure you will need your sunglasses for the ECL exposure !
Good luck with your experiments
Your choice of antibodies is probably more critical. I would recommend those from Cell Signaling Technologies. Also, make sure to use 3% BSA as a blocking agent and not dry milk, since milk has a lot of phosphoproteins.
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