Here is my protocol:
1. Collect small intestine from mice and push out the stool.
2. Flash freeze in liquid nitrogen and then store the tissue at -80C for later use.
3. Homogenize tissues in RIPA buffer from Cell Signal plus inhibitor coctail and sonicate, followed by the incubation for 4h on ice.
4. Spin down and transfer the supernatant into a new tube to determine protein concentration. Adjust to the same concentration.
5. Add SDS sample buffer and load to run the gel.
6. Ponceau stain the membrane after the transfer.
My problems:
When I stain the membrane with ponceau I found the total protein varies from lane to lane even with loading the same quantity of total protein.
And then I go to blot GAPDH to make sure the loading is correct. Attached is the GAPDH signal. I am confused because I never experienced this problem with Western blot. Same loading, different signal.
Can anybody help me figure out what is wrong? I will add more information if required.
I have had similar issues with westerns from tissue extracts. My first recommendation would be not to use ripa as it not not very efficient. I would use 10% glycerol, 2% sds, 50 mM tris pH 6.8. Not protease inhibitors needed due to the high sds which blocks proteases. This will extract pretty much all protein. The lysate may be very viscous due to the genomic dna but just pass it through a needle several times to shear the dna and the viscosity will be reduced.
Also, I would run a protein assay, load a coomassie gel based on this, the adjust the protein loading for the western based on densitometry of the entire lane from the coomassie gel.
Hope this helps.
Mark
I suggest you to do protein quantification one more time and try to probe with actin because the expression of GAPDH gets affected in some cases.
I have had similar issues with westerns from tissue extracts. My first recommendation would be not to use ripa as it not not very efficient. I would use 10% glycerol, 2% sds, 50 mM tris pH 6.8. Not protease inhibitors needed due to the high sds which blocks proteases. This will extract pretty much all protein. The lysate may be very viscous due to the genomic dna but just pass it through a needle several times to shear the dna and the viscosity will be reduced.
Also, I would run a protein assay, load a coomassie gel based on this, the adjust the protein loading for the western based on densitometry of the entire lane from the coomassie gel.
Hope this helps.
Mark
Thank you, Mark. You think lysing is not efficient with RIPA? I did sonication for 15min.
Do you think there is something (protein-containing stool...) contamination, so that I can detect protein concentration but no GAPDH signal in membrane?
The problem seems to be either differential protein loading or differential expression of GAPDH. How are you measuring protein -OD or Bradford etc ? Are you looking at differences in tissue protein expression eg cytokines or in secretions - eg anti-microbial peptides / proteins? Small intestine contains a lot of mucoid material, if you are doing an OD measurement on total extracted material it may absorb at similar ODs as protein (?). Likewise are you doing a DNAse / RNAse digestion to get rid of nucleic acids as well? (this is probably not strictly necessary). When dealing with small intestine I attach a capillary tube to the gut with a syringe at the other end and flush through several times with PBS or media. There is a lot of mucus present which is not obvious initially. Alternatively slice the gut open and rinse thoroughly or bush opened gut with a fine hair paintbrush to remove mucus. If you are freezing tissue in N2 can you grind it up in a mortar and pestle before adding a lysis buffer? I would also try b-actin as another marker of protein
What is the secondary band at the bottom of your picture? Is it a proteolytic cleavage or a non-specific band?. It follows the same intensity as the GAPDH. If it is "non-specific" then it would appear you have an equal loading problem. If it is specific, then you may have an issue with your protease inhibitors.
Ian Teo
I suggest one more ultrasonication before quantification and loading. The homogenization could be one problem.
Thank you all for good ideas. Mark's lysis buffer is a good one. I will try that. And also I will make sure small intestine cleaning up and sonication.
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