Enzyme-linked immunosorbent assay
by Karmella Haynes, 2013
Principle: Proteins are captured on the bottom of a micro well plate, either by direct binding or by a conjugated antibody "trap". A second antibody is added to detect one specific type of protein. A counter-stain antibody (usually HRP-conjugated) is used to generate visible signal, which is proportional to the number of proteins. Normalization (e.g., using the number of cells per lysate sample, and a purified protein with known concentration if you're fortunate to have one available) can be used to calculate proteins per cell.
DIRECT ELISA, HRP DETECTION
This key feature of this approach is the attachment of proteins directly to the bottom surfaces of the micro wells. A specific protein is detected with a primary and secondary-HRP. The advantage over "sandwich" ELISA is that fewer antibodies are needed, but this method is known to be less sensitive/ accurate. Because of this I recommend this method for artificially over-expressed or abundant naturally expressed proteins.
- Well-Coated Amine Binding, 8 well strip plate, clear (G Bioscience #786-753)
- FemtoELISA-HRP kit (G Biosciences #786-110) -- thaw to room temperature
- 10x femto-TBST
- 2x NAP-Blocker
- femtoELISA HRP Substrate
Cell harvesting and counting
- Harvest cells following the standard procedure.
- Resuspend the pelleted cells in 1 mL FACS buffer (1% FBS in 1xPBS) and place them on ice.
- Filter 750 uL of the cells through a strainer cap.
- Transfer 250 uL of cells to a 1.5 mL microfuge tube. Keep on ice.
- Use the remaining 500 uL cells for cell counting by flow cytometry. Set the sampling volume to 20 uL. Calculate cells per uL.
- Transfer 50,000 cells to a 1.5 mL microfuge tube.
- Follow a standard protein prep procedure: http://openwetware.org/wiki/Haynes:ProteinExtraction
- Perform a Bradford Assay to calculate the concentration of total protein in your samples.
- Store the samples at -20°C or continue to the next step.
- Make 1x femto-TBST: Dilute the 10x femto-TBST to 1x in molecular biology-grade water. Make 3.5 mL per sample.
- Wash the wells: Empty the storage buffer from the wells. Wash the wells three times with 200 uL 1x femto-TBST
- Dilute the protein samples to a final concentration of 1 - 20 ug/mL in 1xPBS, final volume of 100 uL.
- Transfer the 100 uL diluted protein samples to the Amine Binding wells.
- Incubate at room temperature for 1 hour.
- Empty the liquid into a waste container. Eliminate residual liquid by tapping the inverted tubes on a paper towel.
- Make 1x NAP-Blocker: Dilute the 2x NAP-Blocker to 1x in 1x femto-TBST. Make 600 uL per sample.
- Blocking: add 200μl of diluted 1x NAP‐Blocker to each well. Incubate at room temperature 30 minutes. Empty the NAP‐Blocker and gently tap out residual liquid onto a paper towel.
- Dilute the primary antibody to a suitable concentration in 100 uL 1x NAP-Blocker (per sample). Add 100 uL diluted antibody to each well.
- Incubate for 1 hour at room temperature.
- Empty the liquid and gently tap out residual liquid onto a paper towel.
- Washing: Fill each well with 1X femto‐TBST (200μl) and wait for 30 seconds then invert the wells to empty and gently tap out the residual liquid from each well. Repeat the washing procedure 4‐5 times.
- Add 100μl HRP‐labeled secondary antibody solution (diluted in 1x NAP‐Blocker) to each well.
- Incubate for 1 hour at room temperature.
- After incubation, empty the plate and gently tap out the residual liquid.
- Washing: Fill each well with 1x femto‐TBST (200μl) and wait for 30 seconds then invert the plate to empty and tap out the residual liquid from each well. Repeat the above washing steps 4‐5 times.
- Finally add 350μl of 1x femto‐TBST into each well and wait for 5 minutes. Tap out the residual wash from each well and plate is ready to develop with femtoELISATM HRP Substrate.
- Add 100μl of femtoELISA HRP Substrate into each well. A soluble blue color develops, which can be read at 370nm or at the 620nm to 650nm range, using femtoELISATM HRP Substrate as a blank.
What to do with your data: calculate unknown protein concentration(s) per cell
- G Biosciences. Well-Coated Amine Bonding 96-well plates. http://www.gbiosciences.com/PDF/Protocol/Well-Coated_Amine_Binding_Plates.pdf
- G Biosciences. femtoELISA-HRP Manual. http://www.gbiosciences.com/PDF/Protocol/femtoELISA-_HRP_Kit.pdf