a. If you hadn’t used blocking solution what might the staining have looked like on your nitrocellulose membranes?
b. If you had boiled the protein extracts prior to the experiment
what effect might this have had on your results?
The expression pattern for the final three cyclins is shown above. The aim of this week’s experiment is to verify how M-cyclin is varying within the synchronised tobacco BY2 cell culture. Protein levels will be assays using a dot blot immunoassay in which a crude protein extract is coated onto a nitrocellulose membrane and probed with specific antibodies. This is a simplified version of the western blot assay, common in cell biology, in which proteins are first separated by molecular weight on a gel, transferred to nitrocellulose membrane and then probed with antibodies.
~ Blocking solution (TBS containing 0.1% Tween 20 and 5% skim milk powder) (20 ml in glass vial).
a) blocking solution is so much important in the western blot because in blocking you block all unoccupied sites on your membrane to prevent the non specific binding of antibodies and other detection agent.if you didn't used background solution then there too much noise in background and it not possible to see beautiful bands of your protein of interest accurately.
b) its depend on different protein samples according to their properties. it is not necessary to boil the sample, sometimes boiling worsen the sample but heating the sample at 90 degree for 5 minutes ( it denature the protein of interest) do not cause any problem and will give required results.
a. If you hadn’t used blocking solution what might the staining have looked like on your...
a. What organisms were the tubulin and the cyclin primary antibodies raised in? b. What was the purpose of the tubulin control? What other proteins might you have used instead of tubulin? The aim of this week’s experiment is to verify how M-cyclin is varying within the synchronised tobacco BY2 cell culture. Protein levels will be assays using a dot blot immunoassay in which a crude protein extract is coated onto a nitrocellulose membrane and probed with specific antibodies. This...
Can you please explain in simple terms the methods of this experiment? I'm having difficulty visualizing and understanding what is being done. I have copied and pasted some of the text from the article. Thank you! Abstract : Obesity, high-fat diets, and subsequent type 2 diabetes (T2DM) are associated with cognitive impairment. Moreover, T2DM increases the risk of Alzheimer's disease (AD) and leads to abnormal elevation of brain beta-amyloid levels, one of the hallmarks of AD. The psychoactive alkaloid caffeine...