western blotting quantification imagej mac

#WESTERN BLOTTING QUANTIFICATION IMAGEJ MAC SOFTWARE#

SDS PAGE are mainly used for the estimation of protein size and protein purity, determination of protein subunits, monitoring protein integrity, Comparison of the polypeptide and different samples and Post-electrophoresis applications, such as Western blotting. The length of the SDS-Protein complex is proportional to the molecular weight of the protein and is separated on the basis of their size. Under these conditions, all reduced polypeptide bind SDS and the SDS-Protein complex forms a rod with negatively charge and similar charged density. In SDS-PAGE, the protein mixture is denatured by heating at 100☌ in the presence of excess SDS and a reducing reagent (β-mercaptoethanol) is employed to break disulfide bonds. SDS Polyacrylamide Gel Electrophoresis (PAGE) is a common type of electrophoretic technique useful for estimating the molecular weight of proteins. The rate of migration of samples through the electrical field depends on the net charge of the protein/nucleic acid, the size and shape of the molecules, and also on the ionic strength, viscosity, and temperature of the medium through which the molecules are moving.

The basic principle of all electrophoretic procedure is that the charged molecules in aqueous solution will migrate in an electric field. To compare the density of bands on an polyacrylamide gel using online software.Įlectrophoresis is a technique used to separate and purify macromolecules such as proteins and nucleic acids on the basis of the differences in their size, charge or conformation. In the end, the integrity and robustness of the published scientific data should be part of the considerations while planning image analysis experiments.In this experiment, Image processing techniques are applied to compare the density of bands on an polyacrylamide gel quantify features on a sample image when automated lane and band analysis is not appropriate or possible, such as in dot blots. Sometimes, a qualitative interpretation might be better suitable than a flawed quantification. IMHO: Not because we can measure everything means also we should do it.

#WESTERN BLOTTING QUANTIFICATION IMAGEJ MAC SOFTWARE#

I can only recommend to use either the software of the Western Blot imaging system in the lab or alternatively GelAnalyzer because it allows to stick pretty well to the recommended procedure by Western Blot material suppliers (which I would guess are the most experienced people in that particular field) and as seen in the publication above. Hammond, “A defined methodology for reliable quantification of Western blot data.,” Mol. So, generally there are many pitfalls related to WB measurements.īesides the literature list in one of the linked posts above, mainly the following gives a good insight into the procedure: And band selections which overshoot the actual band also lead to wrong results.

One cannot reliably quantify bands by making boxes around them and using analyze measure nor do horizontal lines fulfill the requirements. Here is one video which is explaining a little more detailed the considerations of WB in general, normalization as well as measurements The latter and pretty much of most other ones completely ignore every thing mentioned in scientific literature regarding Western Blot measurements (and the pre-requisites for it). There are many videos online like the linked one above. But I cannot contain myself to add my 2 cents to this topic, because some of those videos make me like… Image capture software and Image formats. The housekeeping protein taken here is -tubulin. It exhibits three lanes and four proteins as A, B, C and D. No offense to no one making those videos or taking them as orientation in case of the lack of other available resources. The western blots are taken from Aquatic Ecology and Fish Biology Laboratory, Department of Zoology, Visva-Bharati University. This was the one I looked at Analysing blots and gels with ImageJ/Fiji - YouTube

Western blotting quantification imagej mac