Protein extraction is an important step for Western Blotting in cancer research.
The proteins from samples (cancer cell line) need to be extracted efficiently without degradation.
Although the protocol of this protein extraction technique is quite scientific and technical, I still wish to share as maybe someone who is going to do the cancer research will find it useful.
I am going to explain the nuclear and cytoplasmic protein extraction technique.
Before we start to do the protein extraction, we need to prepare the reagents and the equipments.
There are 3 reagents (CER I, CER II and NER) that we need in nuclear and cytoplasmic protein extraction.
Before the reagents can be used, we have to add protease and phosphatase inhibitor cocktails to these 3 reagents.
The protease and phosphatase inhibitor cocktails provide the solution with full sample protection to protect the protein from degradation during the extraction.
In my cancer research, I add 1:100 ratios of protease and phosphatase inhibitor cocktails to the reagents.
As I need 200 µl of CER I for each sample to be extracted, I have to add 2 µl of protease and phosphatase inhibitor cocktails to mix with the reagent.
Now we can start the protocol of nuclear and cytoplasmic protein extraction.
1.
Firstly, we have to spin down 20 µl of cells into a 1.
5 ml microcentrifuge tube at 500 xg for 3 minutes.
After spinning, the supernatant was discarded using a pipette.
2.
Then, we have to add 200 µl of ice cold CER I to the cell pellet.
The mixture is vortex vigorously for 15 seconds so that the pellet is fully resuspended.
The tube is incubated on ice for 10 minutes.
3.
Consequently, we have to add 11 µl of ice cold CER II and vortex again for 5 seconds.
Then, the tube is incubated on ice again for 1 minutes and vortex for 5 seconds too.
4.
Next, we need to centrifuge the mixture.
Centrifugation is the process to separate the contents in the mixture.
After the centrifugation at 16,000 xg for 5 minutes, we need to transfer the supernatant which is the cytoplasmic extract to a new pre-chilled tube.
Just keep the tube on ice until used.
5.
Then, we need to extract the nuclear protein.
The insoluble pellet is resuspended with 100 µl of ice cold NER and vortex for 15 seconds.
6.
The sample is put on the ice for 10 minutes and vortex for 15 seconds.
This step is repeated 4 times for a total of 40 minutes.
7.
Later, the sample is centrifuged again at 16,000 xg for 10 minutes.
The supernatant which is nuclear extract is transferred to a clean pre-chilled tube and keep it on ice until used.
The nuclear and cytoplasmic protein extraction for Western Blotting in cancer research is done.
After the protein extraction, we need to continue with the quantification of protein concentration by using Bradford assay before we can proceed to the SDS-PAGE.
The proteins from samples (cancer cell line) need to be extracted efficiently without degradation.
Although the protocol of this protein extraction technique is quite scientific and technical, I still wish to share as maybe someone who is going to do the cancer research will find it useful.
I am going to explain the nuclear and cytoplasmic protein extraction technique.
Before we start to do the protein extraction, we need to prepare the reagents and the equipments.
There are 3 reagents (CER I, CER II and NER) that we need in nuclear and cytoplasmic protein extraction.
Before the reagents can be used, we have to add protease and phosphatase inhibitor cocktails to these 3 reagents.
The protease and phosphatase inhibitor cocktails provide the solution with full sample protection to protect the protein from degradation during the extraction.
In my cancer research, I add 1:100 ratios of protease and phosphatase inhibitor cocktails to the reagents.
As I need 200 µl of CER I for each sample to be extracted, I have to add 2 µl of protease and phosphatase inhibitor cocktails to mix with the reagent.
Now we can start the protocol of nuclear and cytoplasmic protein extraction.
1.
Firstly, we have to spin down 20 µl of cells into a 1.
5 ml microcentrifuge tube at 500 xg for 3 minutes.
After spinning, the supernatant was discarded using a pipette.
2.
Then, we have to add 200 µl of ice cold CER I to the cell pellet.
The mixture is vortex vigorously for 15 seconds so that the pellet is fully resuspended.
The tube is incubated on ice for 10 minutes.
3.
Consequently, we have to add 11 µl of ice cold CER II and vortex again for 5 seconds.
Then, the tube is incubated on ice again for 1 minutes and vortex for 5 seconds too.
4.
Next, we need to centrifuge the mixture.
Centrifugation is the process to separate the contents in the mixture.
After the centrifugation at 16,000 xg for 5 minutes, we need to transfer the supernatant which is the cytoplasmic extract to a new pre-chilled tube.
Just keep the tube on ice until used.
5.
Then, we need to extract the nuclear protein.
The insoluble pellet is resuspended with 100 µl of ice cold NER and vortex for 15 seconds.
6.
The sample is put on the ice for 10 minutes and vortex for 15 seconds.
This step is repeated 4 times for a total of 40 minutes.
7.
Later, the sample is centrifuged again at 16,000 xg for 10 minutes.
The supernatant which is nuclear extract is transferred to a clean pre-chilled tube and keep it on ice until used.
The nuclear and cytoplasmic protein extraction for Western Blotting in cancer research is done.
After the protein extraction, we need to continue with the quantification of protein concentration by using Bradford assay before we can proceed to the SDS-PAGE.
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