We want to detect a membrane protein of ~120kD. The student used Mannitol/sucrose buffer for cell lysis - we did not see the expected up-regulation after treatment. However, immunofluorescence with exactly same treatment showed a ~20% increase. Every steps in the western blot was checked and was fine. I am wondering that since it is a protein located on the cytosolic membrane, the lysate buffer might be too gentle? Should we use RIPA instead? What is the advantage and disadvantage of using RIPA versus mannitol/sucrose?
In my experience the best and fastest way to lyse cells when you simply want to see the total content of a protein is to directly lyse them in Laemmli buffer: you will obtain a very viscous lysate, then vortex vigourously and boil for ten minutes. If it is still viscous add more Laemmli buffer and boil again.
Hi Qi,
I would think that the difference you observed with the two methods is due to the sensitivities of the two methods used. Was the difference with immunofluorescence significant?
In general, buffers with detergents (like in RIPA) are needed to release membrane or cyto-skeleton bound protein. However, if you are getting the right band in western, you must have released enough of your target protein to pick up. You could try RIPA on a few samples just for comparison. Cheers.
Hello, I have compared 3 different cell lisis to get my protein which is inside SF9 and H5 cells. 1) containing NONIDET p-40, EDTA, Tris Cl, etc (Lerch 1989), 2) NETbuffer ph( (NaCl, Tris, NaEdta) + Sarkocine and 3) RIPA.. The best one to release my protein (75kDa) was no. 2.
good luck!
In my experience the best and fastest way to lyse cells when you simply want to see the total content of a protein is to directly lyse them in Laemmli buffer: you will obtain a very viscous lysate, then vortex vigourously and boil for ten minutes. If it is still viscous add more Laemmli buffer and boil again.
I agree with Stefania Rigacci that if you want to see the total content of a protein, Lammli buffer may be enough. I also agree with Aniko Huizer-Pajkos or Pajkos that the 20% increase in immunofluorescence may be due to the sensitivity of the method because this increase is not easy to be quantified in WB.
Yeah RIPA buffer would be a good option! Simply, 10% SDS also works fine!!
I generally use Mammalian Protein Extraction Reagent (MPER) +Protease Inhibitor Cocktail(PIC) from Thermo Scientific in the ratio of 100:1.
I recommend RIPA buffer. I used RIPA buffer in my expreinece ( lysis of cancer cell line), and the results was so good.
Thank you all, especially Aniko! I know the 20% increase may not be so great to pick up although IF assay showed significance. So I wanted to use WB as a confirmation, however it has not worked.
I guess we will try RIPA and see if it works. If not, we may switch to flow cytometry?
If you detect your membrane protein in wb, try to decrease the quantities of proteins loaded per lane in the SDS-PAGE, sometime the wb signals are in saturation.
I suggest you to use RIPA buffer gives the lowest background, but can denature some kinases.
I have seen this problem before, and in my cases it was related to the target protein being present in the insoluble fraction of cell lysates, so you do not see an increase by western, but you do by immunocytochemistry. Consequently I used this method. I have posted this method before, but it helped me with a number of proteins. It is based on a very chaotropic lysis buffer called killer buffer; 2%SDS, 2M Urea, 14% sucrose, 1mM Sodium Fluoride,1mM Sodium Orthovanadate, 25mM Beta Glycero phosphate.
1. Remove the tissue culture medium, and rinse with ice cold PBS, incubating the last wash for 2 minutes to chill the samples. (1ml for a 24 well plate, 2ml for a 6 well plate, 12ml for a 90mm petri dish, 30ml for a 150mm petri dish).
2. Place a sheet of absorbant paper on the bench and remove the PBS.
3. Bang the plate or multiwall onto the paper to remove any excess PBS without letting the paper touch the cells.
4. Add killer buffer to the cells (70μl for a 24 well plate, 100μl for a 6 well plate, 200μl for a 90mm petri dish, 400μl for a 150mm petri dish).
5. With a circular motion scrape the lysates around the plate (for the multiwell plates, use a shaft guard pipette with the end bent back, and a cell lifter for the plates) until the lysates is viscous- this is the genomic DNA suddenly released from the nucleus and stripped of all its histones..
6. Place the plate at an angle and gradually scrape the lysates to the lowest edge.
7. Using clean scissors cut the last 3-5mm off the end of the pipette tip at an angle and remove the lysates and transfer it to a Qiashredder column which has been placed in an Ependorff tube.
8. Centrifuge the Qiashredder columns for 2minutes at 6,000 rpm to shred the genomic DNA.
9. De-nature the protein by boiling the lysates at 100C°C for 3minutes.
10. The lysates can be stored on the bench for a while, or at -20°C for longer.
11. Measure the protein concentration using a BCA assay kit.
12. Add 2-Mercaptoethanol to 0.1% and Bromophenol blue to 0.0005% from stocks. The sucrose in the lysis buffer makes the protein extract sufficiently dense to sink in the wells of you gel.
Good luck
@Stefania Rigacci, if Laemmli sample buffer is used to lyse cells, is protease inhibitor cocktail required? 1x buffer or 2x buffer? Can Laemmli buffer protect phospho-protein? Thanks.
I agree with Subhadeep Chakrabarti, usually protease and phospatase inhibitors are not needed when lysing with Laemmli buffer. If the phosphorylation event that you are studying is very transient (i.e. growth factor receptors) wash your cell fastly placing culture plates on ice, then add lysis buffer and boil. If you drain your cells very well before adding laemmli buffer, then 1X is enough. Otherwise use 2x.If you want to determine protein concentration with BCA, use Laemmli buffer without MeSH and bromophenol blue (these can be added to samples before SDS-PAGE)
Some protease inhibitors are small peptides and can affect the results of a BCA assay, but if you measure your BSA standard made up in lysis buffer containing the inhibitors you should be OK, unless your expected protein concentration or very low.
Good luck!
I usually do not add protease inhibitors, since proteases should be promptly inactivated by boiling SDS
@Stefania Rigacci and Subhadeep Chakrabarti, Thanks for your help.
1) Do you mean boiling the 2x Laemmli Sample Buffer (no β-mercaptoethanol and bromophenol blue) to 100oC and adding to cells immediately? If so, do you think 2% SDS (the concentration in 2x sample buffer) alone is also good to lyse cells and protect (phosph-) protein?
2) Did you ever use this 2x sample buffer (no β-mercaptoethanol and bromophenol blue) to homogenize flash frozen tissues?
Usually I rapidly scrape cells in Laemmli Buffer, vortex and then boil. If you want to preserve phosphorylation it is better to place dishes on ice while scrapeing. Sometimes I've added phosphatases inhibitors to buffer, but this does not significantly affects results.
I've never used Laemmli buffer to homogenize tissues, because this will produce a lot of foam....
I have used M-PER Mammalian Protein Extraction Reagent (Thermo) for total cellular protein extraction. Though I have not worked with membrane proteins, this gives a fairly good amount of protein. They also have a Mem-PER reagent for membrane proteins. May be you can try that.
If the many suggestions above do not work out, I would recommend a longer transfer and adding SDS to the transfer buffer while decreasing the amount of methanol in the buffer. For DAGL, also about 120kd, we add 0.1% SDS to the transfer buffer while decreasing the MeOH to 10% from the normal 20%. We also routinely transfer overnight at 4C or maybe 3h on ice.
Hi,
It is okay to use Triton for lysis buffer when working with membrane protein. Also, do you think RIPA will be suitable for this?
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