I am preparing myself for western blot of my samples, but I have confusion in preparing the protein samples because there are a lot of protocols given and I am using RIPA buffer specifically for the same
Hello Priyansha Singh
The first step in sample preparation is isolating proteins from their source. Usually, proteins are isolated from cells or tissues via lysis. The protocol for sample preparation for Western Blot will differ depending on the sample type. If you are using mammalian cells as samples for Western Blot, then you may follow the protocols provided below.
For adherent cells you may follow the following protocol.
1. Remove the culture medium and wash the cells with ice-cold PBS. Place the dish on ice when you perform this step.
2. Aspirate PBS and add ice-cold RIPA lysis buffer (~1 mL for 100 mm plate; ~0.5 mL for 60 mm plate; ~200-400 µL for 6-well culture plate). Agitate cells for 10 mins at 4 degree C. Using cell scrapper, scrap the cells gently off the dish.
3. Collect the lysate and transfer to a microcentrifuge tube. Centrifuge the sample at ~14,000 x g for 15 minutes to pellet the cell debris.
4. Transfer the supernatant to a new microcentrifuge tube and discard the pellet.
For suspension cells you may follow the following protocol.
1. Centrifuge the suspension cells at 2,500 x g for 10 minutes to pellet the suspended cells.
2. Wash the cells once by resuspending the cell pellet in ice-cold PBS. Pellet cells by centrifugation at 2,500 x g for 10 minutes.
3. Add ice-cold RIPA buffer (100ul per cell pellet). Pipette up and down to resuspend the pellet.
4. Shake mixture gently for 10 minutes on ice. Centrifuge at ~14,000 x g for 15 minutes.
5. Transfer supernatant to a new microcentrifuge tube and discard the pellet.
If your sample is a tissue sample then you may follow the following protocol.
1. Dissect the tissue on ice and weigh. Use a ratio of ~50 mg tissue to 1,000 µL of ice-cold RIPA lysis buffer. Use a smaller volume of lysis buffer if a more concentrated protein extract is required.
2. Homogenize the tissue on ice along with the appropriate amount of ice-cold RIPA lysis buffer.
3. Centrifuge the sample at 10,000 × g for 5 minutes to pellet the tissue debris.
4. Transfer supernatant to a new microcentrifuge tube and discard the pellet.
Determine the protein concentration of the lysate prepared from above using the BCA protein assay. Use the appropriate protein concentration of the sample to be loaded on the gel in SDS-PAGE gel electrophoresis. Then proceed with Western Blotting.
Please note: Immediately following cell lysis, proteolysis, dephosphorylation, and denaturation begin to occur. This activity should be kept to a minimum by preparing samples on ice or at 4˚C and by adding protease and phosphatase inhibitors fresh to the RIPA lysis buffer before you begin the lysis process.
Best.
Dear Priyansha Singh
RIPA buffer is a good extraction buffer for most protein types. We are using it in our lab, so I will provide you with the protocol we perform:
1. Add 2% of PI to the desired amount of RIPA buffer.
2. Aspirate the old medium.
3. Wash with ice-cold PBS two times.
4. Add RIPA buffer (20 ul for 24-well plate, 40 ul for 12-well plate ...).
5. Leave it for 3-5 min.
6. Scrape cells with a scraper.
7. Collect the cell lysate and move it to an Eppendorf tube.
8. Incubate on ice up to 20 - 30 min.
9. Homogenize @ 6000 rpm, 30 - 45 s.
10. Heat samples for 5 min @ 95 C.
11. Measure your protein concentration.
12. Preserve the samples @ -20.
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