From my samples, I can get a maximum of 5-10 micrograms of total protein. I need to quantify a protein with low expression. Standard WB does not seem to be the appropriate method. Someone have experience in a similar situation?
Hi,
you can try to enhance your Western Blot signal by using a more sensitive ECL substrate. They are available from all major suppliers and usually named "PLUS".
You could also try to further enhance your signal by using an enhancer kit that should further increase the signal intensity, e.g.:
http://www.millipore.com/catalogue/item/2650#
If you are looking for an alternative to Western Blotting, have you considered ELISA? Normally, you can detect very low amounts of your protein of interest and it is more quantitative than standard Western Blot.
Estimation of total protein content or relative quantitation of a particular protein?
We try to estimate the relative quantitation of a specific protein with expected low expression
Hi,
you can try to enhance your Western Blot signal by using a more sensitive ECL substrate. They are available from all major suppliers and usually named "PLUS".
You could also try to further enhance your signal by using an enhancer kit that should further increase the signal intensity, e.g.:
http://www.millipore.com/catalogue/item/2650#
If you are looking for an alternative to Western Blotting, have you considered ELISA? Normally, you can detect very low amounts of your protein of interest and it is more quantitative than standard Western Blot.
tagging your protein with fluorophore ( e. g. dansylation ) may resolve your problem. Although I never perform this experiment.
I agree with Philipp, if you cannot get it with regular WB and the reagents in the market, maybe you could give it a try to ELISA. You can customize your own ELISA protocol, and if you have got good specific antibodies it should work, at least to check relative expression in different samples.
Just to understand the question, do you have:
1. several micrograms of your proteins in a mixture of proteins and need to quantify your specific protein?
2. or several micrograms of pure protein?
The answer depends on which one it is.
I am not the expert but is the affinity chromatography only effective when the protein you want to separate have a tag ? If not maybe this protein can be separated from big "dilited" samples by affinity chromatography - probably the problem is a need of huge amounts of antibody in that method? Is it possible to relatively quantify the protein from different "diluted" samples by immunoprecipitation or affinity chromatography?
Thanks all your comments
Hi Jeffrey,
We have about 10 micrograms of total proteins from cellular extract and we need to quantify a specific protein suspected is low represented (but present) in our cells. The cells were not cultured cells ( so not tags can be used) are purified cells from a tissue in a window of the development
If you know information like pI and if your protein is stable and purifyable, one approach maybe to try to purify the protein of interest using Anion exchange chromatography. It usually helps to separate the protein from the majority of the contaminating proteins.
Hi! You can also consider dot blot which is the easier way. If you don' t have dot blot facility you can do it manually and using serial dilution of your sample.
If you know exact mass, you can try mass specific ms/ms with known amount of marker protein to know quantity even in picograms
Silver staining is a standard method for detection of protein in low concentration. You may use precipitation (ammonium sulphate/ TCA), ultrafiltration (Centricon) or lyophilization to increase the concentration of your protein and try ECL..
An excellent alternative to colorimetric techniques and to complicated blotting procedures is to measure directly the UV absorption of the peptide bond at 205 nm. For details, please have a look at the 1974 paper by R.K. Scopes in Analytical Biochemistry Vol 59, p 277-282, entitled "Measurement of protein by spectrophotometry at 205 nm". This technique is highly sensitive and does not cause destructions. It is, however, influenced by many substances including some buffer components that absorb light in this range. It is, in my hands, ideal for purified or highly enriched protein preparations.
Good luck!
Maybe you think about the technique of my youth that calculates protein concentration by a very easy and highly sensitive approach based on far UV absorption:
Protein concentration (μg/ml) = 144 (A215nm – A225nm).
This one goes back to the 1950s, published by W.J Waddell.
It works nicely and without destroying anything in your samples, but of course needs the precautions mentioned in the 205 nm procedure, too.
To compare protein expression, the usual way is western / ELISA / mass spec
I am curious to heard any other alternative :)
By the way, immuno histology can also provide really interesting results!
Hi, Jesus, you may try immunolocalization as excellent alternative to WB. This method even give you more information than WB, because you will find which cells and at which developmental stage have a maximum protein level.. Theoretically you can even make a quantitave calculation how much molecules of the proteins have been present in each cells. But, of course, it is not so easy..There are several protocols for immunolocalization. Good luck!
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry