The best negative controls are prepared from knockout

If you are using a recombinant protein, a positive control containing an endogenous form of the target is also a good idea. Positive and negative controls: These controls are essential to prove your results are valid. Why are the bands not showing? Why is the background so high? Why is it always me that hurt? Look at the beautiful results of the friends next to me Xinsheng is envious, so I compiled and summarized how to perform WB quality control and what checkpoints to pay attention to. Where the first detection linear range overlaps, the quantification of proteins in this overlapping region will be good.

However, in the latter, they do not overlap, and it is easy to see how changes in protein concentration affect the band strength of one protein without affecting the band strength of another protein. The best negative controls are prepared from knockout or untreated cell lines. For proteins with low expression of the target protein, low-expression internal parameters such as Lamin B1 and HSP60 can be selected protein. When loading, be sure to add an equal amount of protein sample to ensure a fair comparison to minimize loading errors. Internal control: There are several widely available control antibodies for loading, such as GAPDH, actin or tubulin. In this case, the quantification is inaccurate. In addition, researchers need to demonstrate that the expression of the reference protein does not change due to changes in experimental conditions. Protein samples: After preparing the lysate, use BCA or Bradford and other detection methods to carefully measure the protein concentration.

The biggest challenge here is that the internal control and target protein usually have different linearity in the same sample dilution series, which affects the accuracy of quantification. Don't overload the gel! If your expression goal is very low, you can choose a gel with a large well volume to increase the load. When your positive control fails to signal, you know that the method may be problematic. . This will help clarify any specific issues related to the primary antibody.

Positive controls are usually lysates or tissue extracts with overexpressed proteins.The friends who have done the WB experiment have experienced anxiety during imaging, and they are at a loss when they come out. Due to the high abundance of these proteins, it is very common to see oversaturation of the reference control protein in the expression of samples lab plasticware adapted to low abundance targets. Negative controls are also valuable, often not producing target lysates or tissue extracts

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