Hi Pawan,

RIPA buffer is not ideal if you want to look at proteins associated with the actin cytoskeleton.  I'm assuming you are using Triton-X as your detergent.  If you spin down your cell lystates, the fraction called the Triton-X insoluble fraction will contain cytoskeletal proteins.  Therefore, we use detergents such as CHAPS (10 mM) or NP40 (0.5 to 1%) and do not spin lysates after lysing cells.  By "clearing" your lysate, you are losing valuable proteins (likely the one you are trying to detect). 

After incubating detergent solubilized lysates on ice, we directly add 2x sample buffer without preclearing by centrifugation.  The lysates will be very thick/viscous because of the cellular DNA.  In order to disrupt the DNA in the samples we heat for an additional 5-10 min (up to 10-15 min total) at 90 C.  If the sample is still too thick to load we will reboil the samples until they become less viscous. 

If you are just trying to detect your overexpressed protein, you can lyse your pellet directly in 1x sample buffer.  Again, we don't spin our samples down before loading because of the likelihood of losing proteins. Technically it is not a whole cell lysate if you are removing a fraction or population of proteins by centrifuging your samples.

Although you can't do a protein assay with this method, you can pellet equivalent cell numbers and lyse with the same volume of sample buffer.  This will allow you to load approximately similar amounts of protein between samples. 

if you do not need to do IP or protein conc. from the cell lysate, I would just lyse cells in 2xLeamlies buffer, after 5min at 95C and quick spin, you can do western. this way the lysate should contain all the proteins.

have you tried using detergent free lysis buffer or a mild detergent such as MPER from Pierce?  

Did you do simple vortexing or sonication?......I have seen students facing similar problem in our lab....We overcome this by doing sonication (either in RIPA or EIA buffer) not simple vortexing....Also dissolution of cells pellet directly in 2x protein loading buffer (along with heating at 100 C for 5-10 minutes) works best..... 

Dear All,

Thank for your valuable suggestion. I have some question regarding your suggestions which are as follows:-

a)  I can directly boil the sample in 2X laemmli buffer but I have many samples which I have to do western along with this protein. How should I estimate the protein concentration of all samples to get input same of all without any disparity by western ?

b) No, I haven't tried MPER if you have the recipe it will be easy for me to prepare it by myself and try that.

b) What pulse should I give to clear my lysate without damaging protein content  of the lysate and will it be completely clear so that I can directly estimate the protein concentration by  BCA without involving any high spin steps ?

Looking forward to your response. Thank you in anticipation.

Hi Pawan,

RIPA buffer is not ideal if you want to look at proteins associated with the actin cytoskeleton.  I'm assuming you are using Triton-X as your detergent.  If you spin down your cell lystates, the fraction called the Triton-X insoluble fraction will contain cytoskeletal proteins.  Therefore, we use detergents such as CHAPS (10 mM) or NP40 (0.5 to 1%) and do not spin lysates after lysing cells.  By "clearing" your lysate, you are losing valuable proteins (likely the one you are trying to detect). 

After incubating detergent solubilized lysates on ice, we directly add 2x sample buffer without preclearing by centrifugation.  The lysates will be very thick/viscous because of the cellular DNA.  In order to disrupt the DNA in the samples we heat for an additional 5-10 min (up to 10-15 min total) at 90 C.  If the sample is still too thick to load we will reboil the samples until they become less viscous. 

If you are just trying to detect your overexpressed protein, you can lyse your pellet directly in 1x sample buffer.  Again, we don't spin our samples down before loading because of the likelihood of losing proteins. Technically it is not a whole cell lysate if you are removing a fraction or population of proteins by centrifuging your samples.

Although you can't do a protein assay with this method, you can pellet equivalent cell numbers and lyse with the same volume of sample buffer.  This will allow you to load approximately similar amounts of protein between samples. 

Hi Pawan,

I never worked with the cell line you use. But I know that desired proteins sometimes cannot be recovered from the supernatant after centrifugation because as it happens they get into the fraction of the so-called inclusion bodies as they are maybe not folded naturally. Have you considered this?

Hope it helps and good luck all along...

Difficult, I would personally try separate the cell contents by differential centrifugation and do a western on them. Also a question, did you put SDS in your RIPA buffer when you lyse the cells? If not, you can add SDS to it, it might help.

Yes I have put SDS in the RIPA buffer.

Try the protocol in this paper. Basically you separate the cells into different fractions - detergen (TritonX-100) soluble and insoluble fractions, then do a western on them. You should get something out of this. I've done it before, it's not too difficult but you need to use an ultracentrifuge rather than the bench top one. Hope this helps

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055765/

Thanks for the article.

Hiiii. 

There is way to get cytoskeleton or cytoskeleton enrich fraction. So if you follow the protocol and using special buffer, you could be able to get cytoskeleton fraction and further do experiments as you desire.