The over goal of this western blotting protocol is to Perform western blots on small numbers of cells. This method can help answer key questions in the hematopoietic stem cell field such as the presence or abundance of specific proteins or activation of signaling pathways. The main advantage of this technique is that it requires fewer than 20, 000 cells thus fewer animals are needed to analyze rare populations of primary cells.
We first had the idea for this method when we were trying to find ways to reduce the number of mice we needed to use for hematopoietic stem cell analyses. Demonstrating the procedure will be Xiongwei Cai, a post doc from my laboratory. Use a swinging bucket rotor to centrifuge 1.5 milliliter tubes containing sorted hematopoietic stem and progenitor cells at 500 times gravity for five minutes at four degrees Celsius.
Once the centrifugation is over, discard all but 100 microliters of supernatant, without disturbing the cell pellet. If there are less than 5, 000 cells, and the volume of the sorted sample is less than 0.2 milliliters, first transfer the cells to low binding 0.2 milliliter PCR tubes before centrifugation. However, if there are less than 5, 000 cells, but the volume of the sorted sample is more than 0.2 milliliter, then centrifuge the cells at 500 times gravity for five minutes at four degrees Celsius.
After centrifugation, discard all but 200 microliters of supernatant. Then re suspend the pellet and the remaining 200 microliters of supernatant. Next, transfer the cell suspension to 0.2 milliliter PCR tubes and again, centrifuge.
Then re suspend the pellet in 20 to 30 microliters of supernatant and discard the rest. If necessary, add phosphate buffered saline in 2%fetal bovine serum, or 2%bovine serum albumin to the re suspended cells. Then use an automated cell counter to determine the cellular concentration using two to five microliters of re suspended cells.
Next use a 20 or 100 microliter pipette tip to transfer a volume of 500, 1, 000, and 2, 000 hematopoietic stem and progenitor cells to new 1.5 milliliter tubes. Use a swinging bucket rotor to centrifuge the cells at four degrees Celsius at 500 times gravity for five minutes. Then follow the manufacturers instructions to dissolve phosphatase and proteasome inhibitor and DMSO to prepare a 100 fold stock solution.
Next dilute the four fold Laemmli sample buffer with equivalent volumes of distilled water to make a two fold Laemmli sample buffer. Then add appropriate amounts of the 100 fold stock solution of phosphatase and proteasome inhibitor to adjust the final concentration to two fold inhibitors, and two fold Laemmli sample buffer. Adjust the volume of residual supernatant in the tube such that adding equal volumes of two fold Laemmli buffer along with the phosphatase and proteasome inhibitor will achieve a final concentration of 500 cells per microliter and one fold Laemmli buffer.
And then re suspend to form the lysate. Before loading the gel, heat the sample lysates at 95 degrees Celsius in a heat block for five minutes. Using standard protocols electrophoresis one to 40 microliters of lysate equivalent to 500 to 20, 000 cells through SDS page at 100 volts.
After treating poly vinylidene membrane with methanol for then seconds wash the membrane with distilled water briefly. Then transfer the protein from the gel to the membrane according to the instructions provided by the manufacturer of the transfer apparatus. After transfer, block the membrane with two milliliters of 2%bovine serum albumin and phosphate buffered saline with 0.1%Tween overnight at four degrees Celsius according to standard protocals.
After blocking the membrane, incubate it with diluted antibodies overnight at four degrees celsius. Then wash the membrane thrice with phosphate buffered saline with 0.1%Tween 20 for five minutes each at room temperature. Next incubate the membrane with two milliliters of enhanced chemiluminescence buffer for one minute at room temperature.
Detect the signals using an imaging system. To compare the beta actin signals lysates were prepared from urine hematopoietic stem and progenitor cells and subjected to western plot analysis. It is clear form the amino blot that there is a decrease in beta actin signal from the lysates prepared from 2, 000 to 500 hematopoietic progenitor and stem cells loaded in 1.5 millimeter wells on a 12%SDS page gel.
Further, to compare the beta actin signals obtained from different well sizes cast on the gel. Lysates were loaded in 1.5 and three millimeter wells on the gel and then subjected to western blot analysis. The blot shows that the beta actin signal obtained from the lysate prepared from 500 hematopoietic progenitors loaded in a 1.5 millimeter well is much stronger than when loaded in a three millimeter well.
Next, to compare the signals for eucaryotic initiation factor 4G, actin and phosphorylation of ribosomal protein S6 in response to cytokine stem cell factor stimulation the lysates obtained were analyzed by amino blot. The blot shows the presence of stronger signal intensities for all three proteins in the hematopoietic re genitors with cytokine stem cell factor stimulation. In comparison to the control in vitro it is also clear that there is and increasing signal intensity for all three proteins from different hematopoietic stem cell volumes.
Once mastered this technically can be done in 48 hours. If it is approached properly. While attempting this procedure it's important to remember to accurately count the cells and to direct the lysate cells with a loading buffer.
After this development it is technically pelleted away for the researchers in the field of stem cell research to explore signal pathways in small number of cells. After watching this video you should have a good understanding of how to do western plotting with a small number of address pieces. Be careful with the regions you're using for SDS page.
Thank you for watching. Good luck with your experiments.
