ELISA blocking/testing HRP and not working - I cannot seem to be able to see HRP binding (Dec/08/2010 )

Hi All,

I am doing an exercise in order to see whether my HRP is binding to the wells and the strenght of the signal but it doesn't seem to be working.
Here is the protocol I am following (as given by my supervisor):

1) I am using 3 columns, column 1 has BSA, column 2 Tween and column 3 is my control.
2) I am using a 10%BSA stock and a 10%Tween stock, from these, I make 10 series dilutions (10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 and for the last well water, so 0).
3) I coat the first strip with 320ul of each BSA dilutions and I coat the second strip with 320ul of each Tween dilutions.
4) I then incubate for 1 hour at RT.
5) I wash twice with PBS
6) I add 30ul of HRP (1:2000 dilution)
7) Incubate for 35 minutes
8) Wash 3 x 5 minutes with PBS
9) Add 100ul of TMB

For the control column:

1) The first 3 wells are washed with water
2) 30ul of HRP added to each well but the last 2
3) Incubation of 35 minutes
4) Washed 3x5 minutes with PBS
5) Add 30ul to the last 2 wells
6) Add 100ul of TMB to the whole stripe

Now, this is meant to be an exercise and I am supposed to see a gradient from bottom to top.
The bottom should be darker as there is less blocking agent (BSA and TWEEN very diluted).
The top ones are meant to be very blocked as these wells had higher concentration - so I wouldn't expect a signal there.

Ok, I am 100% sure my HRP works (because I have just tested).
Inhibition test works as well and I see a gradient but the blocking does not work.
As well, when done today I can see my control strip works but columns 1 and 2 (with the BSA and TWEEN) do not seem to show much colour....I can of slightly see something at the bottom but is not consistent as my last well with water at the end of column 1 and 2 should turn blue but what I get is a very mild sky blue for 1 and for the other one I get nothing....therefore as these are just waters and have been done at the same time....I think it could be 1) human error, way I pipette, wash, etc or 2) a problem with my BSA and TWEEN stocks (which are a bit old and nor prepared by me).

The last 2 strips in my control - the 2 wells with just HRP and TMB work as they are very blue and turn green --- so there is a strong signal so I guess my HRP is not sticking? why do the controls work though? is the PBS the problem? HRO is diluted in PBS though and is fine.....

Well, any thoughts and help from anyone that perhaps has done this more than me is highly appreciated.

First, clarification. You are not doing a 'binding' test nor is there any 'inhibition'.

Do you have ‘results’ from others who have run the same ‘test’? This would be a first step to check on the procedure….

You are basically seeing how much sticking you have of an enzyme to wells coated with either BSA or tween. Actual coating is usually 100-150 ul/well…and blocking is done right to the top. You are seeing a trend but very little color and your enzyme and substrate are active. Additionally, you are starting with 100 mg/ml and going down to 1 ug/ml for the BSA; usually sufficient blocking is seen at 1-2% BSA/PBS. I have never blocked with such a low level of BSA and Tween alone is almost never used for blocking but for either washing or to reduce non specific binding in assays. I do not know if Tween-20 remains stuck to plastic wells…it is usually proteins that adsorb.

I do not know how much color to expect at the 1 ug/ml ‘blocking’. One quick thing you can do is to check the OD of the stock BSA and confirm the protein concentration.

You mentioned your ‘technique’. Any carry over of the bsa will block wells.