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Capture AB (1) Dilute capture antibody (here; anti-IL-12p40/p70) in 0.1 M NaHCO3 (pH 8.2) and add 50 ul to appropriate # of wells of Costar EIA medium bind plates. [in our case, we use the first 2 rows for our standards, and the rest of the rows (here just 1) for our samples. So We would need 12 x 3 x 50ul = 1800 ul. So we should make about 2 ml of this solution.] Incubate at 4C for overnight or in the incubator for 2 hrs. (2) Wash with nanopure water 1x. Pat Dry. Blocking Buffer(3) Add 150 ul Blocking Buffer ("BB"). Incubate 60 min at 37 [BB is 3% BSA, 0.05% Tween 20 all in PBS. BSA is a powder. When you hear %, always think of # g in 100 ml. So 3% would be 3g BSA in 100 ml PBS. .05% Tween = .0005 x 100 = .05 ml or 50 ul Tween 20.] [In our particular example, we are using 36 wells x 150 ul = 5400 ul or about 6 ml. So for BSA, 3% would be .03(6)= .18 g BSA. .05% Tween would be .0005(6) = .003 ml or 3 ul] (4) Remove. Pat Dry. Standard and Samples(5) The first 2 rows of the plate will contain our standard in this experiment. So 1st add 50 ul Dilution Buffer to each well in the 1st 2 rows and then do a 1:2 serial dilution of our standard IL-12 in triplicate. [note sometimes dilution buffer will be the same as blocking buffer, sometimes not! In this experiment, we use a lower concentration or 0.5% BSA. So again when here percents, think of 100 ml! (if you will be using g, you can take your % based on 100, but if you are using liquid it would be 0.5 ml up to 100ml) So will need .5 g in 100 ml. Here, because will need quite a bit, you can make the full 100 ml] Our standard in this case is a stock concentration of IL-12 from the freezer of 100 ng/ml. In this experiment, we will do everything in triplicate. The first 3 wells of the 1st row will contain just the 50 ul DB ("empty wells"). The next 3 wells will contain a desired concentration of 50 ng/ml. So we can just take our 100 ng/ml stock and add to our 50 ul buffer (already in the well) to get a starting concentration of 50 ng/ml. Then mix this and take 50 ul of it and it to the next 3 wells to get a concentration of 25 ng/ml and down the rod for 12.5 ng/ml, etc. This is a 1:2 serial dilution in triplicate for rows 1 & 2] Add 50 ul of our samples in duplicate to row 3. Incuate at 37C for 60-120 min(6) wash with nanopure water 2 x. Pat Dry Detection AB(7) Add 50 ul of biotin-anti-Il-12 (2 ug/ml)diluted in DB and incubate 37C for 60 min
Sample EILSA Protocol for IL-12 p40/p70 [Again, detection AB should be
prepared for the experiment. We would need 12 x 3 x 50ul = 1800 ul. So we
should make about 2 ml of this solution. Our stock concentration in this
experiment says .5 mg/ml. We want a concentration of 2 ug/ml. So how much should
we take out of our stock tube to make up 2 ml? 1st change the stock
solution to the appropriate units. .5 (8) Wash with nanopure water 2x and pat dry. (9) Add 50 ul of streptavidin-HRP (1:1000). Place at 37 C for 30 min [this means we can make up the streptavidin-HRP by taking 1 ul and adding 1000 ul buffer] (10) wash with nanopure water 4-5x. This wash is very important. Pat dry (11) Add 50 ul of TMB Substrate (Signma one-step TMB (T-8540) and allow the color to develop at Rm Temp (generally 5-30 min) (12) Add 50 ul of Stop (1N H2SO4). Read at 450 nm. [You can use Excel to generate the mean of the triplicate and duplicate data above. Excel can also generate the slope and y intercept from this data. You can use the equation "Reading-Y intercept/slope" to come up with the various concentrations and hence line for our sample data.] |
