Corrections: at 5:19, Linear scale should be linear range. At 7:47: it's not the Bromophenol Blue that increases the density of the sample to sink into the gel, it's the glycerol in the sample buffer.
Hey my life saver,...i have a question...can you please elaborate why estimation is needed for running western blot in an aspect of same amount of protein loading in every well for control and treatment samples!?
So that you can be sure that if you see one band is stronger or less than the other, you know that it is real biological difference, rather than being due to you not adding the same amount. The loading control confirms that you loaded equal proteins to begin with.
you are saying that if you load 30ug of proteins into each lane, you can't have a straight line (linear), and thus, you can't use housekeeping genes like B-actin for protein normalization?
Yes, that's right: with 30ug or more, you have so much protein there, that your B-actin or other housekeeping protein, would be too saturated to detect differences.
The linear range is where you can accurately measure input differences, because this is the concentration at which the analyte concentration (i.e. housekeeping gene) is linearly proportional to the input amount (i.e. protein loaded). You want to stay below 30ug of protein because it’s the input amount where you have a constant slope. You want to avoid concentrations (i.e above 30ug) where the response is not-constant i.e. not proportional to amount of protein input. Hope this is clearer :)
The Journal of Biological Chemistry (JBC) recommends that Total Protein staining be used for Western Blot Normalisation: www.jbc.org/article/S0021-9258(20)39480-1/fulltex
LOL! I appreciate the feedback. I will try to make future videos clearer. Was there any question you had in particular that I could try to answer for you?
Corrections: at 5:19, Linear scale should be linear range. At 7:47: it's not the Bromophenol Blue that increases the density of the sample to sink into the gel, it's the glycerol in the sample buffer.
Thank you
Hey my life saver,...i have a question...can you please elaborate why estimation is needed for running western blot in an aspect of same amount of protein loading in every well for control and treatment samples!?
You estimate the proteins so that you can load the same amount of protein (usually ug amounts: e.g. 20ug)
@@adwoabiotech thats what i asked why we need to add same amount of protein in each well and what is loading control ?
So that you can be sure that if you see one band is stronger or less than the other, you know that it is real biological difference, rather than being due to you not adding the same amount.
The loading control confirms that you loaded equal proteins to begin with.
you are saying that if you load 30ug of proteins into each lane, you can't have a straight line (linear), and thus, you can't use housekeeping genes like B-actin for protein normalization?
Yes, that's right: with 30ug or more, you have so much protein there, that your B-actin or other housekeeping protein, would be too saturated to detect differences.
@@adwoabiotechplease could you explain that in more detail? As the only purpose of B-actin is to normalise your loading.
The linear range is where you can accurately measure input differences, because this is the concentration at which the analyte concentration (i.e. housekeeping gene) is linearly proportional to the input amount (i.e. protein loaded). You want to stay below 30ug of protein because it’s the input amount where you have a constant slope. You want to avoid concentrations (i.e above 30ug) where the response is not-constant i.e. not proportional to amount of protein input. Hope this is clearer :)
The Journal of Biological Chemistry (JBC) recommends that Total Protein staining be used for Western Blot Normalisation: www.jbc.org/article/S0021-9258(20)39480-1/fulltex
B
bullshit, not helpfull at all
LOL! I appreciate the feedback. I will try to make future videos clearer. Was there any question you had in particular that I could try to answer for you?