I am trying to confirm my miRNA target by western blotting. I have a ds mimic and a ss inhibitor of my miRNA. Is it necessary to use negative controls for mimic and inhibitor? If yes, how can I interpret the role of them?
Hello Narges Daneshafrooz,
Controls are a necessary component of Western blotting. Use a positive control to ensure that the antibody recognizes the targeted protein and that your protocol is set up correctly. Manufacturers will usually supply a positive control lysate with a primary antibody or specify what type of sample to use as a control. As a negative control, load your loading buffer without a sample to very immunodetection and non-specific binding does not occur.
It’s also necessary to control the amount of sample you load in each lane of the gel and the transfer of the sample from the gel to the membrane. This is called the loading control. Loading controls are a key part of studies comparing the expression level of proteins in different samples. Common loading controls include Beta Actin, ERK, GAPDH, and Tubulin.
References:
1. McNamee MG. (1989) Isolation and characterization of cell membranes. Biotechniques 7(5):466-75.
2. Findlay JBC, Evans WH. (1987) Biological Membranes: A Practical Approach. IRL Press, Oxford.
3. Harlow E, and Lane D. (1999) Using Antibodies. Cold Spring Harbor Laboratory Press, New York.
4. Hames BD and Rickwood D. (1998) Gel Electrophoresis of Proteins: A Practical Approach 3rd Edition, Practical Approach Series, Oxford University Press.
You need at least one positive control and at least one negative control for the target protein. Consistent with a positive result, indicating that the target protein is still expressed, and consistent with a negative result, indicating that the target is silenced
Greeting
We use negative control to be sure that the band that we see is not a fake and accidental one. So, I believe that it is neccessary.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA