Hi All,
I want harvest SH-SY5Y cell and collect the protein sample to do western blot. There are many available method for detaching the cell, such as trypsinization, scraping in RIPA buffer, and PBS-EDTA. I would like to ask you guys which one is the best to get most of the protein.
Another question is "What is the best way to homogiize cell lysate before centrifuge?"
I read some answers in this forum, they mention a method to use needle and syringe to resuspend cell lysate instead of using sonicated homogenization. They wrote that this helps breaking DNA from supernatant. But when i tried it on a small volume of cell lysate about 100 uL, I seems lost some volume in the syringe and create lot of bubbles. Do you know how to do this in proper way?
Thank you so much
Cheerio,
In my hands, scraping in RIPA worked the best. Also because trypsinization might change your results, especially when looking at phospho-proteins.
Syringe homogenization might not be the best choice when using small volumes. Also depends on how well you want your lysate to be homogenized. For my purpose, pipetting up and down a bit harshly usually does the job.
Good luck,
Cindy
In case you need to homogenize your sample you can use small pistills for grinding your lysate in aneppi. But mostly as Cindy already said pipetting and the detergents in Ripa should work.
Regards
Sven
When harvesting cells for protein production or analysis I would always go for scraping. Naturally, when working with membrane proteins Trypsin is a no-go anyway. Can't recommend syringe homogenisation, apart from the issues mentioned above, in our hands results were quite diverse.
As the previous posters have said, scraping on ice with cold RIPA buffer is the best and easiest way to extract protein from cells for routine western blotting. There shouldn't be a need to pass the lysate through a syringe and needle as most of the hard work of cell lysis is done by the SDS and deoxycholate in the buffer itself. DNA contamination is usually not a problem from cells particularly since you will only load a fraction of the lysate for western blot but DNA can be precipitated out by increasing the salt concentration of the buffer, however this might affect your protein of interest.
From my experience scraping of cells in ice-cold PBS, then pellet down cells and lyse cells in ripa buffer always gives me high protein content and good results for western blotting.
However, you may have a problem in diluting your samples rather than the way of collecting cells, or may be you start with low number of cells. For example, in most of the cells I dealt with, After scraping cells cultured on 100mm dish, with 80-90% cell confluence, I dissolve the pellet into 70 ul ripa buffer. Then lysis by pipetting 10 times with 200ul pipette tip (narrow tip) and incubation on ice for 1-2 h. This would give you high total protein content.
another thing is that the specific protein that you are looking at may be its expression is low in your particular cell type. in this case you may need to look at other type of cells that have high expression of your protein of interested, or using transfected cells would be better in this case.
good luck
I agree with previous comments. Vortexing 5-10" your sample before centrifugation may also help solubilizing your proteins.
I found that the best outcome is by using the following procedure:
1. put the plate on ice.
2. aspirate the medium and wash the plate X 2 with ice-cold pbs.
3. add 2ml ice cold PBS and scrape the cells in it, collect the cells into 15ml tube on ice.
4. add another 2ml ice-cold PBS to the plate and collect all of the left-over cells.
5. centrifuge and discard suppernatant.
6. Add ~100ul to 10cm plate ice-cold Ripa containing protease inhibitor coctail, and leave on ice for 30 min.
7. perform 3-4 cycles of freeze-thaw 1 min each followed by vortexing after each thawing ( freeze with liquidize Nitrogen and thaw in 37oC bath.
8. centrifuge for 20 min at 4oC at max speed .
9. collect suppernatant.
Mechanical bead beating (like the Bullet Blender) does a great job of breaking up both tissues and cells harvested in a small amount (less than 200 ul) of RIPA. Then sonicate the sample tube in a sonicator bath (not a probe) to thin the sample and break up the DNA; this combination will prevent loading "gummy" samples on your gels that tend to smear (and gives me the best protein quants) and you don't have to open the tube after adding the beads until the sample has been centrifuged to clear it, so no loss of sample.
Jason's & Loqman's methods have always worked well in my hands. The deoxycholate SDS buffer has been around forever but be careful with the inhibitor cocktail they always give me a screaming headache and perform all the steps on ice. Any form of mechanical disruption works but pipetting is as good as any.
If you would like to prepare total cell lysate, you may lyse the cells directly on culture dish by adding ice-cold RIPA buffer.
i would like to add my experience as i usually work with astrocytes culture and and prepare cell lysate for different experiment. in our lab usually for RNA preparation we use RLT buffer from QIAGEN company and for preparation of western blot we usually use RIPA buffer.
I am doing a time course and seeded cells on a 6 well plate. The cells are adherent. Hence, I couldn't leave the plate on ice since I didn't want cells in the other wells to be affected. I added ice-cold RIPA and scrapped my cells for a couple of min, into eppendorf and then centrifuged for 10 min at 4 degrees C. Would my protein be fine? Would I have any protein? It's a week long experiment.
I see this is old but - prior to conducting your real experiment you could run a test with a few different extraction protocols and/or with a few altered variables just using your control cells of interest to see what produces the best yield or the best resolution for your protein of interest by WB, in your hands. I always like running a test experiment prior to a real one, especially if it is the first time, if you are using reagents you didn't just purchase, or if it's a new antibody you're unsure works...
Regardless of how routine WB is, it can be pretty tricky because of all the variables so I find it best to optimize first before going through the trouble of a real experiment with precious samples.
Also, I have successfully used a vortex with a foam tube adaptor placed at 4C for ~5-10 mins for homogenization of multiple samples simultaneously, and to thin-out the jelly-like consistency from cultured cell lysates.
Best of luck,
Brenda
I have been working with Raw 264.7 cells. I tried to isolate cells from plate using RIPA lysis buffer for performing real time PCR, but it creates a lots of bubbles and the isolation becomes more troublesome. Is there any way to make a cell lysis without making bubble? If I use RIPA lysis buffer, how long do I need to keep it for completing the isolation? Thank you.
After washing cells with ice-cold-PBS, directly add ice-cold RIPA buffer on ice. Lyse the cells on rocker or orbital shaker for 30 min. Please make sure to handle cell lysate gently. Excessive and rough pipetting or vortexing should be avoided to reduce bubble formation.
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