Hello All,
I am planning to collect nuclear protein and cytosolic protein from the same cell sample, and I need to find protein extraction protocol. The cytosolic protein is needed to do Co-IP.
I searched on google and found out a method to extract nuclear protein from cytosolic protein by using just RIPA and NP40/TritonX100 lysis buffers. They mentions that use NP40 lysis buffer first to lyse cells then collect supernatant which is cytosolic protein extract. Then lyses the remaining pellet with RIPA buffer again to collect supernatant, which is nuclear protein extract.
Have anyone ever tried this method? Does it get a good result? Do you know what the centrifuge speed and incubating time with lysis buffer (time period to leave cell in lysis buffer with gentle mixing) in each step? and no need to sonication, right?
Both lysis buffers are home-made and the recipes are:
- TritonX-100 lysis buffer (25 mM Tris•HCl pH 7.4, 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 5% glycerol)
- RIPA lysis buffer (25 mM Tris•HCl pH 7.4, 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 5% glycerol)
Thank you so much
Cheerios,
Hi Asa Wu,
I use the following protocol routinely and it works wonders:
Cytoplasm Lysis buffer (CLB)
50 mM Tris-HCl, pH 7.4
10mM NaCl
0.5% NP-40
0.25% Triton X-100
1mM EDTA
Nucleus Lysis Buffer
50 mM Tris-HCl, pH 7.4
100 mM NaCl
1% NP-40
0.1% SDS
0.5% sodium deoxycholate
1. Collect cell pellet.
2. Re-suspend in cold 1ml CLB with pipette first using 1ml tip, and then adding 10ul tip on top of the 1ml tip. (if using brain, then either use mortar and liquid nitrogen or a 1ml homogeniser).
3. Rotate in cold room for 5 minutes.
4. Spin the supernatant at 4°C, 3000 x g for 5 minutes.
5. Collect supernatant and use it for step 6. Collect the pellet and use it for step 7.
6. Spin supernatant at 4°C, 10000 x g for 10 minutes. Collect supernatant, add 200ul of 0.5% SDS, 0.25% sodium deoxycholate, 0.5M NaCl .This will be cytoplasmic fraction.
7. Re-suspend the pellet in 1ml of Nucleus Lysis Buffer. Sonicate (20sec On/10Sec Off; 60-70% Power;2 Cycles). Spin at 10000g for 20 minutes. Collect supernatant. This will be nuclear fraction.
Good Luck!
Utkarsh
Hi Asa Wu
If you send the email to express for seeking more convenient services and better protocol recently,
I indicated that selleckchem.com the leading of the selleck chemicals (inhibitors) will offer a free protocol file list as to Nuclear/Cytosolic Fractionation Kit.
good luck
sunyi
RIPA lysis buffer (25 mM Tris•HCl pH 7.4, 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 5% glycerol)
plus PMSF(add before use) 1mM, protein inhibitor cocktail 100ul/ml
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