I was trying to make a RIPA buffer to isolate total protein from about 10 M SH-SY5Y cells seeded onto a 150 mm-dish. For that pırpose, I mixed 50 mM Trsi-HCl (pH:7.4), 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA and added necessary amount of water. I completed the buffer just before use by adding 0.2 mM sodium-orthovanadate, 1 mM PMSF, 1 tablet of Roche PhosStop and 1x Sigma protease inhibitor cocktail. During isolation, I added 1.5 mL of buffer and scraped the cells on ice, transferred into two microcentrifuge tubes (was very viscous), and centrifuged at 14,000 rpm for 15 minutes at 4 oC. However, there was no pellet at the bottom, instead, a sticky snot-like structure (probably DNA caused) in solution, and I could not take the supernatant. Also, there was some tiny particles in supernatant, and thought that might be cell debris. Is there any problem with RIPA buffer ingredients, or amount to add, and how can I ensure about its purity If I have a chance to separate supernatant from that sticky structure? Can you give me some suggestions?
Bobbi - this topic has been discussed elsewhere on RG and can be found with a simple search. Sonication puts an immense amount of energy into a sample homogenate via cavitation, a result of conversion of kinetic energy from the vibration of the probe to heat. This subsequently heats the contents of the bubble to extremely high temperatures. So no matter how cold you can get your sample, ultrasonic vibrations produce a heat gradient from the probe. This can create amyloid-like aggregates of various sizes, degrade proteins, disrupt antibody epitopes, alter protein phosphorylation and interactions (even in the presence of inhibitors), generate free radicals, etc. In addition, it can generate variations in sample yield.
I assume you're sonicating to shear DNA/chromatin and avoid the snot-like formation which is likely a mixture of DNA and lipids. DNAse will do this effectively if you continue to find viscous material in your samples. If you're sonicating as a means of physical membrane lysis, I would suggest using more refined chemical methods such as subcellular fractionation (as Attila mentioned). Homogenizing cells or tissue in buffers with proper pH and osmotic concentrain can lyse different cellular compartments. This results in better sample quality and gives you with specific information about protein expression/enzyme function at subcellular levels.
Again, to each his/her own.
Or change a less harsh buffer to isolate nucleus-free cytosolic fraction. Depends on target maybe.
Hi Eray-
Can you answer a few questions?
1) Did you place the samples on a rotator at 4degC after scraping and before centrifuging? Sometimes, for proper lysis with aqueous buffers, they need a little time.
2) What is your target protein?
3) What is the reason you chose to use RIPA buffer?
Hi,
I would try something like this:
1. Incubate cells 2 min lysis buffer
2. Use cell scraper
3. Place the lysed cells on microcentrifuge tubes.
4. Incubate on ice for 30 min.
5. Centrifuge @ 10.000 x g for 10 min @ 4°C.
This has worked for a variety of cells and proteins.
Hope it helps you
I had the same problem with RIPA buffer. Are you washing the cell with PBS before adding the lysis buffer?
The only way I could solve it was sonication. I made sure my lysis buffer was cold, cell pellets were on ice throughout and very brief sonication got rid of the sticky substance.
Hope this helps.
IMO, sonicating SHSY5Y protein samples is not a great idea (for many reasons) unless you're really having problems with signal detection.
Grant-- can you provide the reasons your should not sonicate SHSY5Y samples- we do this (and for other cell types) routinely in the lab to prepare for western blotting and this works well.
Srimoye -- I agree with you, without sonication the mix is too viscous and cannot be fully homogenised.
Incubating for a little longer help a bit - as does adding more buffer (but then you have a too dilute sample).
Good luck !!
Bobbi - this topic has been discussed elsewhere on RG and can be found with a simple search. Sonication puts an immense amount of energy into a sample homogenate via cavitation, a result of conversion of kinetic energy from the vibration of the probe to heat. This subsequently heats the contents of the bubble to extremely high temperatures. So no matter how cold you can get your sample, ultrasonic vibrations produce a heat gradient from the probe. This can create amyloid-like aggregates of various sizes, degrade proteins, disrupt antibody epitopes, alter protein phosphorylation and interactions (even in the presence of inhibitors), generate free radicals, etc. In addition, it can generate variations in sample yield.
I assume you're sonicating to shear DNA/chromatin and avoid the snot-like formation which is likely a mixture of DNA and lipids. DNAse will do this effectively if you continue to find viscous material in your samples. If you're sonicating as a means of physical membrane lysis, I would suggest using more refined chemical methods such as subcellular fractionation (as Attila mentioned). Homogenizing cells or tissue in buffers with proper pH and osmotic concentrain can lyse different cellular compartments. This results in better sample quality and gives you with specific information about protein expression/enzyme function at subcellular levels.
Again, to each his/her own.
I used a similar, if not stronger, buffer today, and I obtained a viscous solution after incubation on ice. After a 10 min spin at 15000rpm, I had a small blackish pellet attached to viscous stuff. I loaded this whole lysate into a 1 mL syringe using a 26G needle. Then, I passed the 600 uL of lysate through a 0.22 um filter. I pushed 1 mL of air into the filter to recover more lysate (final volume = 510 uL). The filtered lysate was clear and had no viscosity.
Guillermo, that's an interesting method to remove DNA.
Did you get reasonable concentration of total protein or your target?
I'm doing western blot with the SH-SY5Y, and l met the same problem.
Actually, I think sonication is not a bad idea if you treat your samples for just about 10 seconds.
And sonication or passing a syringe (Guillermo's way) is recommended in the western protocol on Cell Signalling's website:
http://www.cellsignal.com/common/content/content.jsp?id=western
You should try it.
RIPA always causes altered protein profile and endogenous baseline due to loss of proteins non-proportionally. Strong lysis buffer should be used for complete lysis. The stickiness can be removed by using a spin column with a special filter. Sample can be ready in just a minute.
Melissa - the filter in spin column format is designed to remove the viscosity of the lysate using strong lysis buffer to achieve complete protein profile (true endogenous baseline). The non-systematic protein loss caused by RIPA (a much weaker lysis buffer) is virtually eliminated. Hope this answers your question.
Yes I understand, but I am interested in what exactly you do: Is this column purchased from a company or do you have a lab-based protocol? I understand the purpose; I am trying to understand the practical technique. Thanks!
Melissa - The columns along with different buffers can be purchased from Invent Biotechnologies (www.inventbiotech.com), or Fisher Scientific (you can always call them to add a specific product if it is not on their list yet). There are 18 different kits for protein extraction from different cell/tissues types and species. You can find all technical information on the website.
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