The overall goal of this procedure is to identify key improvements to the classic western blotting technique to produce better resolution, quality, sensitivity, and accuracy of Western data. This is first accomplished by preparing samples with a buffer containing lithium ESAL sulfate to provide the optimal condition for protein reduction and alkylation while preserving protein integrity. The second step is to separate proteins using the BIS tris gel system that completes electrophoresis in 35 minutes.
Next, the eye blot dry blotting system is used to transfer proteins to the membrane in seven minutes. The final step is to detect proteins using infrared fluorescent imaging that utilizes near infrared dyes in two different fluorescent channels to simultaneously visualize two different antigens on the same membrane. Ultimately, these novel modifications of the traditional western blotting method results in more accurate qualitative and quantitative data, while greatly reducing the performance time of this technique.
The main advantages of this technique over the existing western blotting methods is the improved efficacy, consistency, and sensitivity of western data that allows for the accurate quantification of multiple target proteins on the same membrane, while significantly reducing the experimental time of performing this technique. To begin place 15 milliliter conical tubes and near cofluent 10 centimeter square cell culture dishes on ice. Then add five milliliters of ice cold PBS containing five millimolar EDTA to each dish using a cell scraper.
Remove adherent cells from the dish and transfer the cell suspensions into one of the pre chilled 15 milliliter conical tubes. Next, pellet the cell suspensions at four degrees Celsius by low speed centrifugation at 230 GS for five minutes. Once finished, placed the conical tubes on ice and carefully aspirate the supernatant without disturbing the cell pellet.
Wash the pellets with one milliliter of chilled PBS containing five millimolar EDTA, and transfer the cell suspensions to a cold 1.5 milliliter centrifuge tube. Then pellet the cells by spinning the suspension at around 13, 000 Gs for 10 seconds. Following centrifugation, carefully remove the S supernatant without disrupting the pellet and place the tubes on ice.
Next, add fresh protease and phosphate inhibitors at a two x final concentration to Ripa Buffer. Then resuspend the cells at 20 million cells per milliliter in Rippa buffer. Briefly vortex each tube and incubate the suspension for 20 to 30 minutes on ice.
Create a pronuclear fraction by centrifuging the lysates at around 15, 000 Gs for five minutes. At four degrees Celsius, transfer the SNAT to a new tube and place the new tubes on ice. At this point, the nuclear enriched pellet can be discarded.
Then determine the protein concentrations of each sample via BCA. Prepare the samples for electrophoresis using the proportions outlined here. The total volume accounts for pipetting variations with 20 microliters of each sample to be loaded per well.
Next, heat the samples at 70 degrees Celsius for 10 minutes. While the samples are heating, prepare one liter of one XMES running buffer. Once the samples are finished, heating, quick spin the samples and place them on ice.
Then prepare precast bis tress gels in the mini cell unit for electrophoresis. Remove the comb from the precast gels and orient the gel so the shorter cassette of each gel faces inward toward the buffer core. Make sure the gels are level and tightly pressed against the buffer core.
To avoid linkage from the upper buffer chamber, fill the upper buffer chamber with 200 milliliters of one XMES running buffer containing point 25%antioxidant, and then the lower buffer chamber of the mini cell unit with approximately 600 milliliters of one XMES running buffer load two to five microliters of an appropriate ladder into the outside wells, and then 20 microliters of each protein sample into the center wells. Then run the gels at a constant 200 volts for 35 minutes. Watch the output for an expected current of 110 to 125 milliamps per gel at the start and 70 to 80 milliamps per gel at the end.
To begin protein transfer completely separate the mini gels by breaking the bonded sides of the cassette. Discard the shorter plate and carefully transfer the gel from the longer slotted plate into a container with deionized water. Then remove the thick footed portion of the gel located at the bottom.
On rare instances, the gel may attach the shorter plate instead of the longer slotted plate. In this case, discard the longer slotted plate and remove the thick footed portion of the gel located at the bottom. Then carefully transfer the gel from the shorter plate.
Next, assemble the transfer stacks according to the manufacturer's instructions onto the dry blotting device. Carefully remove any bubbles or air trapped between the gel and the membrane to prevent protein band distortion during the transfer. Then select the appropriate transfer program and begin the transfer.
This can take between seven and 10 minutes depending on the size of protein transferred. Prepare the membrane for infrared fluorescent protein detection by blocking the membrane in a minimum of 0.8 milliliters per square centimeter of odyssey blocking buffer for one hour or overnight at four degrees Celsius prior to incubating the membrane in primary antibody. The membrane can be sectioned into various pieces in order to probe for multiple antibodies on the same membrane.
Next, incubate the membrane in primary antibody diluted in odyssey blocking buffer with the addition of 0.1%tween 20 overnight at four degrees Celsius to visualize two different antigens on the same lot, both antibodies must be derived from different host species following incubation with the primary antibody. Wash the membrane three times in TBS with 0.1%tween 20 for 10 minutes with gentle shaking. Then add fluorescently labeled secondary antibodies diluted in odyssey blocking buffer with 0.1%tween 20.
Note the secondary antibodies need to be derived from the same host species as each of the primary antibodies and labeled with different Fluor fours Incubate the membrane in a light protected container for 30 to 60 minutes. Incubating the membrane for longer than one hour may increase background wash the membrane three times for 10 minutes in TBS plus 0.1%tween 20 while keeping the membrane protected from light with gentle shaking, the membrane may be dried or stored in either TBS or PBS without tween 20 at four degrees Celsius until it is scanned and imaged with an infrared imaging system. The fluorescent signal will remain stable for several months if properly protected from light.
This western blot shows the simultaneous detection of total arc one and two in the 800 nanometer green channel and beta actin in the 700 nanometer red channel from Lysates of WM 7 93 human derived melanoma protein. A single channel fluorescent detection in the second panel shows BIM from the same membrane. The two color infrared system can also produce black and white images as seen in the three panels below the quantification of bands of total IR one two beta actin and total BIM was derived from the integrated intensity of each protein band.
The integrated intensity is proportional to the amount of fluorescently labeled secondary antibodies on the membrane. R squared values were used to evaluate the linear regression for each antibody analyzed. WM 7 93 melanoma cells were treated with DMSO or MEK inhibitor for 18 hours.
The two colored Western blot fluorescent detection shows phospho or one two in the 800 nanometer green channel and total IRC one two in the 700 red channel with overlapping phospho irk and total irk signals in yellow. The addition of mech inhibitor results in the loss of phospho irk one two expression yet doesn't affect the total IRC one two expression. The graph on the right shows the quantification of phospho IRC one two normalized to total IRC one two protein levels from the figure on the left.
This was calculated using the integrated intensity of the fluorescent signal and the data is presented as a percent of the DMSO control. After watching this video, you should have a good understanding of how to employ the latest innovative advancements and performing a western blot analysis that will drastically reduce the experimental time and generate higher qualitative and quantitative data.
