Hello. I wanted to prepare whole cell extract for western blotting. Currently, I
Step 1. Lyse cells in detergent (e.g., TritonX-100),
Step 2. Take some lysate for determining total protein amounts (e.g., by Bradford assay),
Step 3. Add SDS sample buffer to the rest of the sample, boil.
However, this method has caused viscosity and made sample loading quite difficult, and the WB membrane showed band streaks upon Ponceau staining. And I think the genomic DNA content that remains in the sample is the reason.
Does anyone know how to effectively remove DNA during preparation of whole cell extract? Thanks very much.
1. Dilute your loading sample with dH2O according to the capacity of your wells.
2. Heat for 10, 15 or 20 min at 95 C if it is suitable to your sample.
3. Add 1% SDS to your lysing buffer, if it does not work add more up to 2%.
1. Take the appropriate amount of cells for your experiment and homogenize them in a buffer containing proteinase inhibitors. 2. Centrifuge the homogenate at 10,000 x g for 10 minutes at 4°C. 3. Discard the supernatant and resuspend the pellet in lysis buffer. 4. Incubate the lysate at 4°C for 15 minutes. 5. Centrifuge the lysate at 10,000 x g for 10 minutes at 4°C. 6. Collect the supernatant (the whole cell extract), and proceed to SDS-PAGE. 7. Dilute the extract in sample buffer containing SDS and boiling it for 5 minutes. 8. Load the samples onto a pre-cast SDS-PAGE gel to separate the proteins according to their size. 9. Run the gel using appropriate running buffer and electrophoresis conditions. 10. Visualize the separated proteins using an appropriate staining method.
Hi Bandla Ramesh thanks a lot for the protocol. I have a few questions.
For step 1. how do you "homogenise" the cells? Does homogenisation here only serve to separate the cells for better lysis later?
For step 3. what is the ingredient in the lysis buffer? I guess for different lysis buffers, we will have different incubation times to allow sufficient lysis (in step 4)?
For step 5. what will be in the pellet after the centrifugation?
And for step 6. why is the supernatant still regarded as the "whole" cell extract even we have discarded whatever that is in the pellet?
Jian Wang in addition to were mentioned by Adam B Shapiro , adding SDS and boiling longer time are key steps if you had already extracted them.
Prepare RIPA buffer containing 1% SDS to lyse cells and then boil 5-10min. Try it.
If you already collect cells in lysis buffer without SDS, add some stock SDS to make final 1%, or 2%.
Order RIPA buffer or prepare by yourself: https://www.cellsignal.com/products/buffers-dyes/ripa-buffer-10x/9806?
Feng Wang Hi thanks for the suggestion! Could I ask why adding SDS during cell lysis can help degrade DNA? What I normally do is add SDS sample buffer to the cell lysate and then boil at 95C for 10mins. What is the advantage of having SDS in the lysis buffer in terms of degrading DNA?
Jian Wang yes adding SDS to the cell lysis buffer can help in degenerating gDNA and lipids, however during cell lysing, homogenization has the highest effect on breaking gDNA.
Of note, you have to use SDS wisely, so it can affect the protein stability, the bands separation and their sharpness.
For immunoblotting from SDS-PAGE gels, try diluting the whole cell lysates 1:50 in SDS-PAGE sample buffer before loading on the gel.
Hej! I would also suggest to dilute your sample quite a lot! We usually lyse the cells in buffer containing detergents like SDS or Triton. Then we centrifuge the sample to remove cell debris. The supernatant is than checked for protein concentration (by Bradford or BCA or such). And for immunoblotting we load a MAXIMUM of 20µg of protein per lane! Depending on your antibody and the abundance of your protein in the lysate much less can be enough!
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