Hi i want to do protein expression study of bacteria at different time and sugar. In this condition what can be the loading control but i can not use GAPDH because i am using different sugars. please suggest something .
Are you expressing recombinant proteins? you can use the cell lysates before induction or cell lysates from non-transformed expression cells as controls. It is also good to include purified and well characterized proteins as positive controls.
I agreed the suggestion of Alemu. for negative control u may use bacterial cell without induction or same bacteria without transformation. for positive control you may use another confirmed transformed cell which protein is well characterise. If u wanted to see the transfer of proteins in membrane then u may use pre-stained colour protein markers.
No its not recombinant. i am using crude total cell lysate of bacteria for bacteria.
Hi Sandeep,
I take it that by "loading control" you mean the detection of a protein that allows you to quantify and compare the amount of sample loaded per lane on a gel?
This is very complicated because almost all proteins will vary with growth phase, growth conditions, temperature etc. GAPDH is usually used but as discussed in this thread on RG, it is not reliable:
https://www.researchgate.net/post/Housekeeping_proteins_loading_controls_for_a_Western_Blot_of_E_coli_cell_lysates2
Most of the respondents recommend total protein stain for such normalisation.
Alternatively if you want to ensure same sample load per well, you could adjust the sample loaded per lane according to OD600 or cell number (determined by microscopy).
Good luck!
If you want to measure the total protein in a gel lane after staining, for instance with Coomassie Blue R, then, using a single known protein for the quantitative standard on the gel may give inaccurate results.
To minimise the error you could use a set of known protein standards as follows.
Obtain proetein molecular weight marker standards that can be supplied by one of the lab supply companies. The total quantity of protein present will be indicated or can be obtained from the supplier and typically there will be 7 different proteins with a range of molecular weights.
Make a solution of these standards in gel sample buffer so that you know the total concentration of protein; you do not need to know the concentration of each individual standard.
Load this standard sample in a series of wells on the gel each with a different quantity of protein but which will cover the range that you expect from your lysate sample.
Load a range of quantities of you lysate sample into another series of wells on your gel.
Run the gel and stain it. Measure the total optical absorbance ( above backgroung) for all of the the proteins in each lane of the gel ( treat each lane separately). Plot a standard curve for the total protein absorbance in each lane containing standard proteins verses total quantity of protein in each lane.
Measure the total absorbance for all the proteins in the lanes contains your lysate samples. Read off this absorbance against the standard curve. This will not be perfect but better than using a single protein.
You could make your own standard proteins mix but this would be more work.
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