Hello All!
I want to test protein expression in 5 different type of muscles (with different morphology) . I have homogenised a varied number of samples per each muscles to increase protein concentration: for example, for muscle A, I took 1 sample, for muscle B I took two samples (due to B being smaller than A) etc.
my question is: when I do the western blot and I load the same amount of 50ug of total protein, will the differential amount of samples of muscles used influence my protein expression?
Furthermore, one of the antibody I will be using should correlate to the amount nicotinic acetylcholine receptors at the neuromuscular junctions (NMJs). Therefore will muscle B express more of this protein than A because I used 2 samples instead of one? Some studies have shown that the density of NMJs does not correlate to the size of the muscle i.e. the bigger muscles sometimes just have bigger NMJs instead of higher amount of NMJs?
Thank you for your help.
Regarding question a) If 1x sample A is from one muscle and the 2x samples B is from one muscle leading to comparable total concentration (50 µg total protein) with different amounts (µl) this CAN NOT influence teh results. (Moreover, expression can not be influenced - since this happens earlier!) I would propose to check equal load with adequate loading controls (e.g. GAPDH or beta-actin expression) and normalize expression of your protein of interest as ratio to this control protein (non influenced by your experimental setting/stimulus).
Regarding question b) There may be differences in total protein detection. However, normalization to an adequate loading controls (e.g. GAPDH etc.) allow to normalize your results and allow to compare sample A vs sample B without any influence of amount or any volume effects in your Western blot experiment.
Once more regarding both questions:
Expression happens in living cells (the muscle). In western blot experiments nothing can influence protein expression. However, your results regarding protein DETECTION may be influenced by differences in total protein amout /maybe concentration or what ever.
To relativize this potential dicrepancies a loading control (houskeeping protein) should be used e.g. a protein which is expressed in a unvarying level (not influenced by the experimental setting/stimulus etc.).
Andreas Eisenreich Sorry, I think my word choice was misleading. What I meant was protein detection. The housekeeping gene I am using is said to be not entirely reliable for muscles (GAPDH) however, it seemed to work quite nicely with my previous experiments. I also have a total protein loading control (Coomassie Staining). I have previously done the experiment from one mouse to reduce any sort of confounding variables in the results. But the antibody I used was hardly detectable that is why I have decided to use 2 mice instead. I am a bit worried as the age of the mice are slightly different and perhaps the expression of the proteins I am investigating might vary from mouse to mouse.
In line with your first question:
No. of samples used will never be proportional to your protein levels on the gel. Since you will be doing a BCA and making sure of loading equal conc. of protein per lane.
Second question :
If you make a lysate from 2 muscle vs 1 muscle. Definitely for both cases even if you load equal conc. of protein on the gel, the probability of getting a sharp band for the former is greater.
Moumita Sarkar thank you for replying. Do you think it will be a problem if the sources of the samples are slightly different: one mouse aged P43 and one P45? I believe they are littermates. So for muscle A, since it’s so big, I need it from only 1 source but for muscle B, since it’s so tiny I need to take it from 2 sources?
Moumita Sarkar with respect to protein detection. Especially for the antibody that is localised at the NMJ? I initially thought that loading an equal amount of protein should give a fair quantification via normalisation of data regardless of amount of samples. But I am now a bit worried that this might influence the detection and give me an outcome that is not truly representative.
In my opinion, age of mice can lead to differences in expected results. Why do you use mice of different ages. Is it possible to use pooled samples from mice of the same age?
If the age is no variable influencing expected results, this parameter can be ignored (e.g. when the goal is to detect the protein itself independed from time effects).
For better understanding:
Please tell me - in brief - what do you want to see in what experimental setting? And what the the goal of this study?
Andreas Eisenreich I want to quantify neurotrophic factors (NTFs) at the NMJ only. Currently my WBs give me total protein in the 5 muscles. I need to ensure that the nAchR antibody is a reliable control to allow me to quantify the NTFs at the NMJ (it should be a good indicator since it binds to NMJ only), hence I need to make sure that the confounding variables influencing the outcome of the AchR antibody are as low as possible. I am using different age mice because 1) these are too small to give me enough concentration from one sample for antibody detection 2) these are just preliminary tests I am doing to find out best controls for quantification. What do you think?
Run the same profile for P43 and P45 and then conclude independently.
Dear Tahmina,
in this case, it is ok to use different sources for total protein from the muscle of different aged mice. In my opinion normalization to constant background expression (e.g. GAPDH) should work and allow to relativize any potential concentration / amount effects (due to sample source etc.).
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