Blot background in AFP

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Aug 6th, 2022
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How to blot background in AFP

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Here are some tips on how to handle and preserve your protein samples for Western blotting: Work quickly. Working quickly can minimize potential damage to your samples by simply allowing less time for them to become degraded. Keep everything cool. Use protease inhibitors.
To reduce high background due to high antibody concentration, titrate the antibodies. A dot blot can be used to titrate both primary and secondary antibodies in a checkerboard like pattern. Blocking the membrane is a crucial step that prevents non-specific binding of antibody to the membrane.
Generally, an AFP level between 0 ng/mL to 10 ng/ml or even up to 40 ng/mL can be normal for adults But normal levels vary, so talk with your healthcare provider about what your results mean for you. An extremely high level of AFP in your bloodgreater than 400 ng/mLcould be a sign of liver tumors.
High background in a Western Blot means a high signal / noise ratio, and it affects the detection of your protein of interest badly. High Background Troubleshooting in Western Blots - Sino Biological sinobiological.com category wb-troubles sinobiological.com category wb-troubles
For a reducing Western blot, samples are dissolved in a Laemmli buffer and then boiled to assure that all the protein in the sample is denatured. They mixture is then rapidly cooled down on ice before loading to keep proteins in their denatured, linear form.
Introduction. Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein. Western Blot: Technique, Theory, and Trouble Shooting - PMC - NCBI nih.gov articles PMC3456489 nih.gov articles PMC3456489
Western Blot possible causes solutions for high background Too much protein per lane. Titrate down the amount of protein loaded per lane. Insufficient blocking of non-specific binding. Adjust blocking conditions.
The study by Harada T, et al. [39] showed that approximately 40% of all cirrhotic patients will have AFP levels higher than 20 ng/ml. An increase in AFP levels (between 10 and 500 ng/ml, and sometimes up to 1000 ng/ml) can be seen in adult patients with hepatitis or cirrhosis with any etiology.
Make sure you use sufficient wash buffer, wash for a long enough time, and agitate the membrane well during wash steps. Any non-specifically bound antibody left on the blot is going to contribute to high background. You may also consider adding additional detergent or changing the detergent in the wash buffer. Help! Why do my Western blots look terrible? - Azure Biosystems azurebiosystems.com blog help-why-do-my-w azurebiosystems.com blog help-why-do-my-w

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