identical results in ELISA assay - I must be doing somthing wrong, but what? (Sep/20/2005 )
QUOTE (Large Mango @ Sep 27 2005, 05:41 AM)
Hi Guys,
I just had a quick look at this and one thing that stands out is that you are using alkaline phosphatase as your detection method.
Inorganic phosphate is a strong competitive inhibitor of alkaline phosphatase so you should use something other than PBS to wash your plates as this can potentially be a big problem. I would use TBS for this instead....
I just had a quick look at this and one thing that stands out is that you are using alkaline phosphatase as your detection method.
Inorganic phosphate is a strong competitive inhibitor of alkaline phosphatase so you should use something other than PBS to wash your plates as this can potentially be a big problem. I would use TBS for this instead....
No I'm sorry but we are doing Elisa assay every day and we use most of the time AP conjugated mAb's without any trouble if we use pBS.
The only problem is the pH of the detection when you add the substrate the pH should be alkaline but for the wash no worries
I would just rinse once with the substrate buffer alone to make sure that the pH is allright before to throw in the substrate mix !
Another thing which you should be very careful is the Na Azide because it blocks the AP enzyme so you should wash well !
Pesji
-pesji-
I've used AP in westerns and we didn't have problems with using PBS-T as a wash buffer or using low % azide in our diluent for primary and secondary abs. Although I suppose the azide would have been washed away by the development step.
Ceri
-Ceri-
I probably should have added that in the Westerns we also did a pre-wash in the alkaline phosphatase substrate buffer before development. I guess this would remove any effect PBS-T may have on the reaction.
-Ceri-
QUOTE (Rosie @ Sep 20 2005, 09:08 AM)
I'm running a normal sandwich ELISA assay in the sequence; Primary ab, 2x wash, block, 2x wash, antigen, 4x wash, secondary ab, 4x wash, detect.
When I detect I'm getting near identical results for every well, including the serial dilutions of my pure protein standards and the alledged blanks (Primary ab and secondary ab but no antigen). However my wells with either no primary ab or no secondary ab added show a blank result.
Any ideas why?
At the moment I'm using a 1xPBS-T wash solution with a PBS wash for the final secondary ab wash (ie. 3x wash plate with secondary ab in PBS-T + 1x wash plate with PBS), a primary ab concentration of 1:2500 and a secondary ab concentration of 1:1500 (ab datasheet for secondary says 1:1000 - 1:2000), and a 3% BSA + sodium azide Blocking buffer (my lable is alkaline phosphatase so that shouldn't be a problem)
Could I be using too much primary ab? Or could it be another problem?
Thanks
Rosie
When I detect I'm getting near identical results for every well, including the serial dilutions of my pure protein standards and the alledged blanks (Primary ab and secondary ab but no antigen). However my wells with either no primary ab or no secondary ab added show a blank result.
Any ideas why?
At the moment I'm using a 1xPBS-T wash solution with a PBS wash for the final secondary ab wash (ie. 3x wash plate with secondary ab in PBS-T + 1x wash plate with PBS), a primary ab concentration of 1:2500 and a secondary ab concentration of 1:1500 (ab datasheet for secondary says 1:1000 - 1:2000), and a 3% BSA + sodium azide Blocking buffer (my lable is alkaline phosphatase so that shouldn't be a problem)
Could I be using too much primary ab? Or could it be another problem?
Thanks
Rosie
Rosie it sound a bit tricky, Which type of secondary antibody are you using is it an anti Mouse ? Anti IgG or anti subclasses ? We need more data to judge where can be the trap !
pesji
-pesji-
QUOTE
No I'm sorry but we are doing Elisa assay every day and we use most of the time AP conjugated mAb's without any trouble if we use pBS.
The only problem is the pH of the detection when you add the substrate the pH should be alkaline but for the wash no worries
I would just rinse once with the substrate buffer alone to make sure that the pH is allright before to throw in the substrate mix !
Another thing which you should be very careful is the Na Azide because it blocks the AP enzyme so you should wash well !
Pesji
The only problem is the pH of the detection when you add the substrate the pH should be alkaline but for the wash no worries
I would just rinse once with the substrate buffer alone to make sure that the pH is allright before to throw in the substrate mix !
Another thing which you should be very careful is the Na Azide because it blocks the AP enzyme so you should wash well !
Pesji
Hmm... The fact remains that Inorganic phosphate is a strong competitive inhibitor of alkaline phosphatase. Why would you want to take the risk when TBS works as well? This is especially true with an ELISA as it is a quantitative tool rather than say a western which is qualitative - semi quantitative at best...
-Large Mango-
QUOTE (pesji @ Sep 28 2005, 04:13 PM)
Rosie it sound a bit tricky, Which type of secondary antibody are you using is it an anti Mouse ? Anti IgG or anti subclasses ? We need more data to judge where can be the trap !
pesji
pesji
The antibodies I'm using are "Rabbit polyclonal to PKC alpha" (primary), and "Goat polyclonal to rabbit IgG (alkaline phosphatase)" (secondary)
Both are from Abcam and both are (aledgidly) tested for use in ELISA assays.
If I can get the PKC alpha to work I will be moving on to a rabbit polyclonal to PKC iota antibody.
Rosie
-Rosie-
QUOTE (Rosie @ Sep 29 2005, 03:24 AM)
QUOTE (pesji @ Sep 28 2005, 04:13 PM)
Rosie it sound a bit tricky, Which type of secondary antibody are you using is it an anti Mouse ? Anti IgG or anti subclasses ? We need more data to judge where can be the trap !
pesji
The antibodies I'm using are "Rabbit polyclonal to PKC alpha" (primary), and "Goat polyclonal to rabbit IgG (alkaline phosphatase)" (secondary)
Both are from Abcam and both are (aledgidly) tested for use in ELISA assays.
If I can get the PKC alpha to work I will be moving on to a rabbit polyclonal to PKC iota antibody.
Rosie
Oops I think I have a slight doubt.
So you coat the plates with your primary antibody which will bind the PKC up to now everything is fine
then you add a Goat anti Rabbit igG and of course it recognize every well of course cause it binds to every primary antibody even if those ones didn't bind any PKC ยจ
I would use a secondary antibody specific for your PKC but recognizing another epitope ! or even the same polyclonal but labelled with AP
Am I clear ? Maybe I don't understand but that's the way I see your problem
You can call me at the instuitute if you want 0041 61 284 8236
Pesji
-pesji-
I get you Pesji! I was wondering about that myself! I'm kind of hopeing it IS that, it will at least be easy to solve.
-Rosie-
QUOTE (Rosie @ Sep 29 2005, 05:14 AM)
I get you Pesji! I was wondering about that myself! I'm kind of hopeing it IS that, it will at least be easy to solve.
Yes Rosie you just have to find another anti PKC antibody rather a polyclonal or you can label yours with a kit
Happy that it helps
pesji
-pesji-