The overall goal of this procedure is to demonstrate the ease of performing a Western blot when using appropriate products. Following sample preparation, proteins are separated based on size and then transferred to a solid phase. The proteins are then probed with antibodies and detected the use of proper protocols and high quality cost-effective reagents produce enhanced western blotting results.
Hi, I'm Jesse Luhan, r and d scientist at EMD Chemicals in San Diego, California. EMD is an affiliate of Merck, KGAA in Dharm stat Germany. Today we're gonna show you a western blotting procedure.
So let's get started. The first step in western blotting is sample preparation. Begin sample preparation by pelleting the cells using low speed centrifugation such as five minutes at 2, 500 times gravity.
Following centrifugation, drain the cell pellets. Well resuspend the cells in cyto buster protein extraction reagent using 150 microliters per 10 to the six cells. Incubate the sample at room temperature for five minutes.
Next, spin the sample for five minutes at 16, 000 times gravity at four degrees Celsius following centrifugation. Transfer the cleared SNA to a fresh tube and proceed with analysis. After the protein concentration of the sample is assessed, prepare the sample for loading the gel to denature the protein.
Use a sample buffer containing an onic denaturing detergent such as sodium doil sulfate or SDS. Boil the mixture at 95 to 100 degrees Celsius for five minutes. Using a commercially available precast gel proteins can quickly be separated based on molecular weight.
Fill the electrophoresis unit with running buffer, which can be made from reagents offered through the Omni PU chemical line. Load the first lane of the well with trail mix protein marker. This marker has three reference bands which allow for monitoring during electrophoresis.
Once the gel is stained, 10 bands are visible, ranging from 10 to 225 kilo Daltons. Load the denatured sample into the remaining wells. Run the gel at 220 volts for one hour.
Once the proteins have separated, stain the gel with kumasi blue to ensure that the proteins have migrated evenly and uniformly using rapid stain and ultrasensitive stain, which is ready to use no DS.Staining is required just as proteins with an electrical charge can be induced to travel through a gel in an electrical field, so the proteins can be transferred in an electrical field from the gel onto a membrane such as NITROCELLULOSE or PVDF. When using PVDF membranes, soak the membrane in methanol for a few minutes, followed by a equilibration in ice cold transfer buffer for five minutes. Also soak the gel for a few minutes in ice cold transfer buffer E equilibration prevents shrinkage of the gel during transfer.
For a wet transfer, assemble a sandwich of the gel and membrane between sponge and paper. Then clamp the assembly together tightly after ensuring no air bubbles have formed between the gel and membrane. Submerge the sandwich in transfer buffer and apply an electrical field.
The negatively charged proteins will travel towards the positively charged electrode. The membrane then stops them, binds them, and prevents them from continuing on. Once the transfer is complete, visualize proteins to ensure even transfer with no air pockets.
This can easily be done with red alert Western blot stain. This stain is ready to use and des staining can be done easily with water. This is the gel once it has been distained by washing in water before the membrane can be proved with antibody, the membrane is blocked.
To prevent nonspecific binding, block the membrane with high quality blocking reagents. To avoid an increased background by incubating it in blocking solution for one hour at room temperature under gentle agitation, then wash the membrane three times in TBST or PBST after incubation. These buffers can be easily and conveniently made using TBS tween tablets and PBS tween tablets respectively.
Once the membrane has been blocked and washed, it can be incubated with the primary antibody. Apply the primary antibody using solution, one of signal boost, which allows the signal to be enhanced significantly. Prepare the primary antibody in solution one and incubate the membrane for one hour.
There is no need to add further blocking agents following incubation. Wash the membrane with TBST. Next, incubate the membrane with the secondary antibody diluted in solution two of signal boost for one hour.
Finally, wash the membrane again several times with TBST for HRP conjugated. Secondary antibodies detection by enhanced chemiluminescence or ECL is traditionally used here. The ECL reagent rapid step is used as it requires no mixing of luminol or enhancer.
Simply spray the membrane a few times, incubate for two minutes, and develop it using x-ray film. Rapid Step offers low picogram sensitivity as well as lighter mission for up to two hours after the addition of substrate. Trail mix protein markers allows for protein tracking during electrophoresis when compared with standard Western blot protocols.
Signal Boost allows for greater dynamic range and sensitivity. Finally, superior sensitivity can be obtained by using Rapid step instead of traditional ECL reagents. We've just shown you how to perform Western blinding efficiently and cost-effectively with a comprehensive range of products.
Remember the quality of your results rely on the quality of your reagents. That's it. Thank you for watching and good luck with your experiments.
