Western blot failure experience summary
Western blot failure experience summary I have done WB for a long time, the biggest difficulty is the sample preparation! Sometimes the degradation of the protein is unimaginable, it may be surprisingly bad, or it may be completely okay. If you do WB alone, it feels more effective to use the principle of "fast and complete lysis, denaturation and preservation". Try to choose a strong enough The lysis solution even lyses the loading buffer directly, then part of the protein is taken out for quantification, and the rest is added to the loading buffer at 100 degrees for full denaturation and then stored separately. Sometimes it is more effective than protease inhibitors.
Another feeling is that the amount of target protein in the sample is limited to the detection limit of WB. In general, the amount of target protein should not be less than 1ng (although the lower limit of the ECL method is 0.1ng, that is, 100pg), preferably more than 10ng. This requires that the potential abundance of the target protein must be fully considered before doing WB. It is best to fully prepare before designing the amount of cells or tissue. Do n’t make the sample thinner, otherwise it will be difficult to handle.
Finally, read more literature. Before making a certain protein, it is best to see how others do it, any special requirements, how to choose internal controls, and so on.
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