ELISA troubling shooting - (Nov/21/2005 )
Hello everyone,
I used commercialized sandwich ELISA plates to determine relative protein levels for my transgenic trees. I tested on 600 samples, and it seemed to work fine. But suddently when I switched to a differnt genotype, I started to have problems. I will really need your help and advice regarding this matter. Here are the problems: I have samples taken from three different years. I didn't have problem with samples taken from year 2004, but very low readings for a majority of samples taken from year 2003 and year 2005(close to 0) even I ran samples from three different years side by side. I extracted new proteins and ran again, and I might get a normal readings for few of them, but the rest of samples stayed almost same. I used bradford method to determine total protein concentration, and used the same amount of total proteins for ELISA. any ideas what might caused the problem, and guidelines for trouble shooting?
Thanks very much for your help!
-Rain
-Jingyi-
QUOTE (Jingyi @ Nov 22 2005, 12:59 AM)
Hello everyone,
I used commercialized sandwich ELISA plates to determine relative protein levels for my transgenic trees. I tested on 600 samples, and it seemed to work fine. But suddently when I switched to a differnt genotype, I started to have problems. I will really need your help and advice regarding this matter. Here are the problems: I have samples taken from three different years. I didn't have problem with samples taken from year 2004, but very low readings for a majority of samples taken from year 2003 and year 2005(close to 0) even I ran samples from three different years side by side. I extracted new proteins and ran again, and I might get a normal readings for few of them, but the rest of samples stayed almost same. I used bradford method to determine total protein concentration, and used the same amount of total proteins for ELISA. any ideas what might caused the problem, and guidelines for trouble shooting?
Thanks very much for your help!
-Rain
I used commercialized sandwich ELISA plates to determine relative protein levels for my transgenic trees. I tested on 600 samples, and it seemed to work fine. But suddently when I switched to a differnt genotype, I started to have problems. I will really need your help and advice regarding this matter. Here are the problems: I have samples taken from three different years. I didn't have problem with samples taken from year 2004, but very low readings for a majority of samples taken from year 2003 and year 2005(close to 0) even I ran samples from three different years side by side. I extracted new proteins and ran again, and I might get a normal readings for few of them, but the rest of samples stayed almost same. I used bradford method to determine total protein concentration, and used the same amount of total proteins for ELISA. any ideas what might caused the problem, and guidelines for trouble shooting?
Thanks very much for your help!
-Rain
hi,
i could not understand exactly what you do(initially you said when you switch genotype, ELISA did not work, in your problems you explained about sampels from 3 different years).
anyway i can suggest you some thing,
that is devide your whole experiment in to two parts
1, experiment part
2, sample part
in experimental part, you make sure that your ELISA works properly every time. this you can achieve by using controls common for all assays which will give you general idea about the variability in your assay. if your positive control every time gives similer results which means sandwich ELISA is working fine, then you have to take care of second part.
in sample part, you said you are looking for relative protein levels of transgenic plants from different years. if you say that all plants merely should show same levels of protein then
i would doubt the storage conditions.
if you make sure that storage is done by same person with same protocol. then you shold look in to the protein quantity(protein of your interest) differences in transgenic plants. in this case you will correlate your experiemtntal sample reading with the positvie control reading.
when you have positive control, then it make sense to compare samples from different years.
i hope this explanation might help you to find hints about your problems.
bye
gud luck
sravan k.payeli
-payeli-
QUOTE (payeli @ Nov 22 2005, 01:42 AM)
QUOTE (Jingyi @ Nov 22 2005, 12:59 AM)
Hello everyone,
I used commercialized sandwich ELISA plates to determine relative protein levels for my transgenic trees. I tested on 600 samples, and it seemed to work fine. But suddently when I switched to a differnt genotype, I started to have problems. I will really need your help and advice regarding this matter. Here are the problems: I have samples taken from three different years. I didn't have problem with samples taken from year 2004, but very low readings for a majority of samples taken from year 2003 and year 2005(close to 0) even I ran samples from three different years side by side. I extracted new proteins and ran again, and I might get a normal readings for few of them, but the rest of samples stayed almost same. I used bradford method to determine total protein concentration, and used the same amount of total proteins for ELISA. any ideas what might caused the problem, and guidelines for trouble shooting?
Thanks very much for your help!
-Rain
hi,
i could not understand exactly what you do(initially you said when you switch genotype, ELISA did not work, in your problems you explained about sampels from 3 different years).
anyway i can suggest you some thing,
that is devide your whole experiment in to two parts
1, experiment part
2, sample part
in experimental part, you make sure that your ELISA works properly every time. this you can achieve by using controls common for all assays which will give you general idea about the variability in your assay. if your positive control every time gives similer results which means sandwich ELISA is working fine, then you have to take care of second part.
in sample part, you said you are looking for relative protein levels of transgenic plants from different years. if you say that all plants merely should show same levels of protein then
i would doubt the storage conditions.
if you make sure that storage is done by same person with same protocol. then you shold look in to the protein quantity(protein of your interest) differences in transgenic plants. in this case you will correlate your experiemtntal sample reading with the positvie control reading.
when you have positive control, then it make sense to compare samples from different years.
i hope this explanation might help you to find hints about your problems.
bye
gud luck
sravan k.payeli
Hi Sravan,
The purpose of my work was to look at the change of transgenic expresson over years. I have two different species to look at. ELISA worked well for one specie, but I started to have problems with another specie as I stated in my previous message. I always have one positive sample run on each plates and it was pretty consistent among plates. So my thinking was something was wrong with samples. I first thought that extracted proteins got degraded, so I reextracted proteins and run ELISA again, and it didn't help much. I used the same amout of total proteins for the all samples, and I expected that I would get pretty close readings for the samples taken from the same tree over three years. But most samples taken from year 2003 and year 2005 were close to background levels, while readings for the ones taken from year 2004 had a nice range. Further suggestions and help will be appreciated. Sorry if I confused you again!
Thanks
Rain
-Jingyi-