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General

My sample lysates were made in RIPA buffer. Can I test these samples with your ELISA kits?

Yes, you can. As you can see below, the composition of the traditional 1X RIPA buffer is very similar to that of our Cell Extraction Buffer (Cat. No. FNN0011). Cat. No. FNN0011 is frequently used to prepare lysates for testing with our ELISA and Luminex™ kits. Our NP-40–based Cell Extraction Buffer (Cat. No. FNN0021) is also used. We recommend diluting lysates made with Cat. No. FNN0011 at least 10-fold in order to lower the SDS concentration to ≤0.01% (v/v) before adding the samples to the ELISA or Luminex™ assay. This recommendation also applies to RIPA lysates prepared in the RIPA buffer shown below. Note that there are other RIPA buffer formulations available that differ to some extent from the one shown here.

Cat. No. FNN0011

1X RIPA Buffer

Cat. No. FNN0021

10 mM Tris buffer, pH 7.4 

10 mM Tris buffer, pH 7.2 

50 mM Tris buffer, pH 7.4

100 mM NaCl

150 mM NaCl

250 mM NaCl

1 mM EDTA

5 mM EDTA

5 mM EDTA

1 mM EGTA

1 mM EGTA

 

1 mM NaF

1 mM β-glycerophosphate or
10 mM pNPP

50 mM NaF

20 mM Na4P2O7 

2.5 mM Na4P2O7

 

2 mM Na3VO4 

2 mM Na3VO4 

1 mM Na3VO4 

1% Triton™ X-100

1% Triton X-100

1% NP-40

10% glycerol

1 μg/mL leupeptin

 

0.1% SDS

0.1% SDS

 

0.05% sodium deoxycholate

1% sodium deoxycholate

0.02% NaN3

Note: Cat. No. FNN0011 and FNN0021 and RIPA buffer should be supplemented with 1 mM PMSF and a protease inhibitor cocktail just prior to use. For the PMSF addition, we recommend making a 0.3 M stock in DMSO, and adding sufficient volume for a final concentration of 1 mM (i.e., 17 μL per 5 mL of buffer). Note that PMSF is very labile and must be added just prior to use, even if it has been added previously. For the protease inhibitor cocktail addition, we recommend Cat. No. 87786 with EDTA, which is a 100X solution in DMSO. The instructions suggest adding 50 μL per 5 mL of buffer. Note that the stability of protease inhibitor–supplemented Cat. No. FNN0011 is 24 hours at 4°C. 

I ran out of the streptavidin-HRP that came in your ELISA (or Antibody Pair) kit. Can I get more?

No. The exact same streptavidin-HRP conjugate supplied in these kits is not available as a stand-alone product. However, it is derived from our ELISA-grade streptavidin-HRP (Cat. No. SNN2004), which we do sell. Remember that the streptavidin-HRP provided in each lot of ELISA or Antibody Pair kits is also lot-specific. So, if you use Cat. No. SNN2004 or another source of streptavidin-HRP, you will have to determine which dilution of SNN2004 works best for you.

I bought one of your Antibody Pair ELISA kits, but there were no buffers included. How should I set up and run the ELISA?

Our Antibody Pair kits include matched, pre-titered and fully optimized coating and detection antibodies, 3 vials of recombinant protein standard, the streptavidin-HRP conjugate concentrate, and a lot-specific technical data sheet. The buffers are not included. The Buffer Kit needed to run the Antibody Pair ELISA must be purchased separately (Cat. No. CNB0011). Note that the 5X Assay Buffer supplied in Cat. No. CNB0011 is used to block your ELISA plates and as a diluent for the standards and samples.

The A450 values that I got for the standard curve in your ELISA kit are lower than the example values shown in the product manual. Why?

There are 2 main causes of poor ELISA standard curves. First, the recommended method for solubilizing the kit standard may not have been followed. The standard should be reconstituted according to the directions indicated on the label, using the standard diluent provided in the kit. No other diluent should be used. The vial should then be swirled or mixed gently and then allowed to sit for 10 minutes at room temperature to ensure complete solubilization. This concentrated standard solution should be used within 1 hour of reconstitution. Also, it should be mixed gently again before preparing the dilutions in standard diluent according to the instructions provided in the product manual. Leftover standard can usually be stored frozen in small aliquots, unless specified otherwise in the product manual.

The second common reason for poor standard curves is that the HRP conjugate was not diluted correctly. The 100X HRP conjugate solution contains 50% glycerol, which makes it very viscous and difficult to pipet accurately. Here is what we suggest to solve this problem: First, let the vial of HRP conjugate come to room temperature. Then, stir it gently with a clean pipet tip to make sure that it is homogeneous. Use only the separate HRP conjugate diluent provided in the kit to dilute it, and follow the dilution instructions provided in the manual. 

The key to diluting the HRP conjugate is to make sure that it is pipetted correctly. You should test that your pipettor accurately aspirates and dispenses the volume of the conjugate-glycerol mixture that is required. If possible, this pipettor should be calibrated so it is accurate and reliable. When you aspirate the viscous conjugate solution, it may take 5–10 seconds for the desired amount to enter the pipet tip. Before transferring the conjugate to the appropriate HRP diluent, make sure that the outside of the pipet tip is dry by wiping it with a lab tissue (e.g., Kimwipes™ tissue), taking care to ensure that the contents inside the tip do not get absorbed by the tissue. Pipet the conjugate into the diluent, and then rinse out the tip by pipetting up and down several times. It is important to get every last bit of conjugate out of the tip. Next, seal the container and mix it gently but thoroughly by rocking it or turning it upside down. This is crucial because the glycerol carries the conjugate quickly down to the bottom of the tube. If the diluted conjugate is not mixed adequately, the concentration of the HRP conjugate will not be what is required. 

Once the HRP conjugate is diluted and mixed gently but well, use it within 15 minutes. Remember that the HRP conjugate diluent is the only acceptable diluent for the HRP conjugate. The diluted HRP conjugate should not be saved because the HRP activity is labile, and it should never be stored and reused.

After I added the chromogen reagent to the plate, I incubated the ELISA as suggested in the manual, but the A450 of the highest standard was higher than what my plate reader can read. What should I do?

Our customers use a wide variety of plate readers. Some of these can’t read absorbances higher than 2 AUFS (Absorbance Units Full Scale), while others can’t go beyond 3 AUFS, for example. If you read your ELISA plates after 30 minutes of incubation at room temperature and 1 or 2 A450 values are off-scale, you can shorten the incubation time. For example, some customers find that 20 minutes is the ideal incubation time because the ambient temperature in their lab is higher than ~72°F (22°C). In this case, higher temperatures increase the rate of the HRP-driven ELISA. Conversely, if the A450 values you get are not high enough, you can increase the incubation time accordingly.

I am getting an elevated background in my ELISA. What should I do?

Here are possible causes and solutions:

Cause

Solution

Insufficient washing and/or draining of wells after washing. Residual solution containing anti–rabbit IgG HRP or streptavidin-HRP can elevate the background if left in the well.

 

 

Wash according to the protocol. Verify the function of the automated plate washer. At the end of each washing step, invert the plate on absorbent tissue on the countertop and allow it to completely drain, tapping forcefully if necessary to remove residual fluid.

 

Chromogen exposed to light prior to use, resulting in a blue color.

Keep chromogen in its vial until you are ready to dispense it into the plate, and then pour it into a reservoir to prevent contamination of the vial with equipment. Do not cover the plate with foil.

 

Incubation time is too long or incubation temperature is too high.

 

Reduce incubation time and/or temperature.

Contamination of pipette, dispensing reservoir, or substrate solution with anti–rabbit IgG HRP or streptavidin-HRP.

Do not use chromogen that appears blue prior to dispensing onto the plate. Obtain a new vial of chromogen.

 

Blanks that have been set up improperly.

Follow the protocol when designating blank wells. Blank wells contain only chromogen and stop solution. Subtract blank well results from all other wells.

 

Incorrect dilution of standard stock solution or standards diluted in serum, culture supernatant, or other.

Follow the protocol instructions regarding dilution of the standard. Dilute standards only in the Standard Diluent Buffer provided in the kit.

 

I have an elevated optical density (OD) reading for my ELISA standard. What went wrong?

Here are possible causes and solutions:

Cause

Solution

 

Incorrect dilution of the anti–rabbit IgG HRP or

streptavidin-HRP working solution.

Warm the solution of anti–rabbit IgG HRP or streptavidin-HRP (100X) to room temperature, draw it up slowly, and wipe the tip with a laboratory tissue (e.g., Kimwipe™ tissue) to remove the excess. Dilute only in the HRP diluent provided.

 

Incubation times extended.

 

Follow incubation times outlined in the protocol.

Incubations performed at 37°C.

Perform incubations at room temperature (~25 ± 2°C) when instructed in the protocol.

 

I see very weak to no color development after my ELISA. What happened?

Here are possible causes and solutions:

Cause

Solution

 

Reagents not at room temperature (~25 ± 2°C) at start of assay.

 

Allow all reagents to warm to room temperature prior to commencing the assay.

Incorrect storage of components, e.g., not stored at 2–8°C.

Store all components exactly as directed in the protocol and on labels.

 

Anti–rabbit IgG HRP or streptavidin-HRP working solution made more than 15 minutes before use in assay.

Use the diluted anti–rabbit IgG HRP or streptavidin-HRP within 15 minutes of dilution.

 

Expired reagents.

Check expiration dates upon receipt of kit and use the kit prior to expiration.

 

Plate read at incorrect wavelength.

The correct wavelength to read ELISAs using the TMB substrate is 450 nm.

 

TMB solution lost activity.

Ensure that the TMB solution is clear before it is dispensed into the plate wells. A blue color and/or the presence of particulate matter indicate that the product is contaminated. Please contact Technical Support if this problem is noted. To avoid contamination, we recommend that the quantity required for an assay be dispensed into a previously unused disposable trough for pipetting. Discard any TMB solution left in the trough and do not put it back in the bottle. Avoid contact between the TMB solution and items containing metal ions.  Do not cover your plates with aluminum foil or aluminum-coated Mylar™ sheets because this can cause color development in the absence of HRP.

 

Attempt to measure analyte in a matrix for which the ELISA assay is not optimized.

Contact Technical Support when using alternative sample types.

 

Wells have been scratched with pipette tip or washing tips.

Use caution when dispensing into and aspirating out of microwells.

 

Incorrect chromogen or stop solution used.

Use only the chromogen and stop solution supplied with the kit.

 

Standard diluent buffer added to all wells rather than the designated wells.

Follow the protocol and only add the standard diluent to the designated wells and to the samples where it is required, or to samples producing signals greater than that of the highest standard.

 

Use of buffer containing azide, which is not compatible with HRP.

 

Avoid the use of azide in the assay.

I got a poor standard curve after my ELISA. Why is this?

Here are possible causes and solutions:

Cause

Solution

 

Improper preparation of standard stock solution.

Dilute the lyophilized standard as directed on the vial label, only with the standard diluent buffer or a diluent that most closely matches the matrix of your sample.

 

Reagents (lyophilized standard, standard diluent buffer, etc.) from different kits, with either different analytes or different lot numbers, were substituted.

 

Never substitute any components from another kit.

 

Errors in pipetting the standard or in subsequent steps.

Always dispense into wells quickly and in the same order. Do not touch the pipette tips on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device.

 

I performed an ELISA assay, and the A450 readings for my duplicate wells were very inconsistent. What could have gone wrong?

Here are possible causes and solutions:

Cause

Solution

Errors in pipetting the standards or samples or in subsequent steps.

Always dispense into wells quickly and in the same order. Do not touch the pipette tip on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device. Check for any leaks in the pipette tip.

 

Repetitive use of tips for several samples or different reagents.

Use fresh tips for each sample or reagent transfer.

 

Wells have been scratched with the pipette tip or washing tips.

Use caution when dispensing into and aspirating out of microwells.

 

Liquid transferred from well to well during incubations.

Adjust the orbital shaker or check for correct rotator rpm. Peel the adhesive plate cover off carefully.

 

Incorrect volumes of materials dispensed into the microwells.

Follow the protocol for dispensing volumes of reagents. Check calibration of the pipettes.

 

Standard diluted with the serum, culture medium, or other buffer.

Dilute the standard with the standard diluent buffer provided in the kit.

 

Particulates or precipitates present in the samples.

Remove any particulates/precipitates by centrifugation prior to dispensing into the assay.

 

Dirty microwells: visible debris within or on bottom of microwells.

Inspect the microwells and invert the plate to remove debris. Wipe the bottom of the plate with an absorbent tissue after each wash step. Never insert tissue into the microwells.

 

“Edge effect” due to uneven temperature between the outer-edge wells and the wells in the center of the plate.

Seal the plate completely with a cover during incubations, and place the plate in the center of the incubator when 37°C incubation is indicated.

 

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