What immuno-method to use? - (Jul/29/2008 )
Dear all,
If you want to detect small amounts of a special protein in human samples, and you have expensive antibodies against that protein, that can not be purchased from any ordinary supplier - what method would you choose for Research and Development (regarding qualitative results, quantitative results, reability, costs, time consumption, adventages and disadventages etc):
ELISA
Western Blot
Dot Blot
Ouchterlony Double Immunodiffusion
Radial Immunodiffusion
I'm grateful for any help in this matter!
/de Winter
What kind of sample? Tissues, cell suspension?
Wound fluid (for example)
Ok you gave us 'wound fluid' as sample matrix. Big question is what is the concentration you wish to detect?
Are we talking pg/ml, ng/ml, ug/ml or mg/ml? Each of these would reflect which method you pursue.
Do you have access to different types of analytical instruments?
Are we talking pg/ml, ng/ml, ug/ml or mg/ml? Each of these would reflect which method you pursue.
Do you have access to different types of analytical instruments?
Well, the concentration IS the big question
. My problem is to try to detect and/or estimate the concentration of an exogenous protein in wound fluid at a given time after application. The protein concentration is most likely very low, but how low is hard to estimate. From mg and lower...I have an HPLC, plate reader, spectrophotometer, maybe sets for SDS-page... I also have antibodies against the given protein.
If the protein level is low, what method shouldn't I use? If it's high, which would be better? Which of the methods is most reliable? Will wound fluid proteins disturb the result even if I have a.b. against the protein, and if so, is one method better than the other in that respect? If all of the equipment and chemicals must be bought, which method would be the cheapest/most expensive? Which is less/most time consuming? And so forth...
I'm really really greatful for all answers!
If you want to detect small amounts of a special protein in human samples, and you have expensive antibodies against that protein, that can not be purchased from any ordinary supplier - what method would you choose for Research and Development (regarding qualitative results, quantitative results, reability, costs, time consumption, adventages and disadventages etc):
ELISA
Western Blot
Dot Blot
Ouchterlony Double Immunodiffusion
Radial Immunodiffusion
I'm grateful for any help in this matter!
/de Winter
I think radial immunodiffusion and ouchterlony double immunodiffusion are outdated methods.
Both methods use large amount of samples and antibodies.
I think sandwich ELISA or Western blot depend on the concentration of proteins in the human samples.
Hope this may help.
Both methods use large amount of samples and antibodies.
I think sandwich ELISA or Western blot depend on the concentration of proteins in the human samples.
Hope this may help.
Reading about RID I got interested by the possibilities, but also scared abot the amount of a.b. I guessed would be needed. Do anyone know the detection limit of RID and approx how much a.b. that must be used?
Would either ELISA or WB be preferred if there are small amounts of protein to be detected? Do they have different detection limits?
Thanksabunch
...and another question!
(I know WB separates the proteins in the procedure) but do I need to seperate my protein from other proteins in the wound fluid in any of the mentioned methods, or do I use my samples directly in all methods?
Please check the following link
for beginner in western blot
http://www.abcam.com/index.html?pageconfig...e&rid=11375
for ELISA
http://www.abcam.com/index.html?pageconfig...e&rid=11422
Hope this may help.
for beginner in western blot
http://www.abcam.com/index.html?pageconfig...e&rid=11375
for ELISA
http://www.abcam.com/index.html?pageconfig...e&rid=11422
Hope this may help.
Yes, that helped me with some of my questions. Thank you!