If you have done SDS-PAGE and Western blotting experiments, you will definitely see curved or blurred bands, unlike the perfect strip shapes shown on your antibody data sheet. Although these curved strips are not attractive in their own right, they affect the molecular weight, which may affect the analysis of strip density measurements, especially when using automated procedures or analyzing proteins with similar molecular weights.
Don't worry, usually you can take five very simple steps to make your perfect imprint appear again.
Cleaning laboratory equipment
It took you a few weeks to train your rare cells, treat them with expensive new compounds, carefully collect and dissolve your samples, and then place your gel between two dirty glass plates. . So make sure your glass plates and combs are clean before casting, which is a very important step to ensure that you are casting a perfect gel.
Shake and stir
A major problem with gel casting is the improper mixing of reagents, especially acrylamide. In order to ensure uniform protein separation in the gel, it is necessary to ensure a constant percentage of acrylamide. But again, you should use the glue as soon as possible for the experiment. A thorough mixing of the gel reagent, followed by an equally important degassing step, gives you a pure gel every time.
Keep reagents fresh
In the same way, the buffer buffers ensure that they are fresh and it is important to adjust to the correct pH. We often find that our western blotting is not repeated. Using a buffer buffer that is not fresh for nearly 6 months, and keeping the buffer in the sun, it may make more sense to throw the buffer away and reconfigure it. At least check the pH of the buffer as it is important for the gel to play an important role in your protein migration.
Low voltage jogging
As mentioned above, we know that you want to get exciting results as quickly as possible. Running your gel at high voltage may cause you to have to dispense the glue and re-run the sample. By running your gel at high voltages, buffers and gels become hot, which is the main cause of your gel warping and bending.
Sampling should be moderate
Reducing strip bending and more about blotting, this step is essential to ensure you get the most accurate results from the blot. Although it is tempting to load the maximum concentration of protein in the largest volume, sometimes reducing the amount of sample will ensure that you get a good band rather than an unrecognizable mark. The use of new antibodies, even new antibody batches, requires testing using the total protein standard curve. This simple step saves you time and samples and makes your analysis faster.
After these five steps, you can make your strips sharp and make them the pride of your lab.
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