Haynes:ELISA

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(DIRECT ELISA)
Current revision (20:31, 2 July 2013) (view source)
(DIRECT ELISA, HRP DETECTION)
 
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==DIRECT ELISA==
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==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.
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.
MATERIALS
MATERIALS
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* Femto ELISA HRP kit (G Biosciences 786-110)
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* 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'''
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** '''femtoELISA HRP Substrate'''
PROCEDURE
PROCEDURE
 +
 +
'''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.
'''Cell lysis'''
'''Cell lysis'''
 +
# Transfer 50,000 cells to a 1.5 mL microfuge tube.
 +
# Standard protein prep procedure.
 +
 +
'''Bradford assay'''
 +
# Perform a [[Haynes:Bradford | Bradford Assay]] to calculate the concentration of total protein in your samples.
 +
# Store the samples at -20°C or continue to the next step.
 +
 +
'''Protein-well binding'''
 +
# 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.
 +
 +
'''Primary antibody'''
 +
# 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. 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.
 +
 +
'''Secondary antibody-HRP'''
 +
# Add 100μl HRP‐labeled secondary antibody solution (diluted in '''1x NAP‐Blocker''') to each well and 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.
 +
 +
'''Signal detection'''
 +
# 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.
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# Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-n).
 
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# Add 500 μL Bradford Reagent to each tube. You will add protein to these later, and ignore the negligible change caused by additional protein volume.
 
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# Add a BSA standard protein solution* to tubes 2 (1μg BSA), 3 (2μg BSA), 4 (4μg BSA), 5 (8μg BSA), and 6 (16μg BSA). (*Note, use the appropriate volume based on the concentration of your stock BSA).
 
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# Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling.
 
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# Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate.
 
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# Do the same for the others, using new wells, but be sure to mix by pipetting up and down before transferring 200 μL of sample to the 96-well plate.
 
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# Use a plate reader to record absorbance at 590 nm (OD 590).
 
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'''What to do with your data: calculate unknown protein concentration(s) per cell'''
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#
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What to do with your data: calculate unknown protein concentration(s)
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REFERENCES
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# Subtract the blank OD 590 value from all other values.
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# G Biosciences. Well-Coated Amine Bonding 96-well plates. http://www.gbiosciences.com/PDF/Protocol/Well-Coated_Amine_Binding_Plates.pdf
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# Plot a standard curve (using Excel) with BSA concentration (x-axis) vs. Absorbance at 590 nm (y-axis). See [http://openwetware.org/wiki/Image:KAH_012510_chart1.tif this example].
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# G Biosciences. femtoELISA-HRP Manual. http://www.gbiosciences.com/PDF/Protocol/femtoELISA-_HRP_Kit.pdf
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# Add a '''line''' of best fit (not a curve) and display the equation.
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# Solve the equation for ''x''. Substitute ''y'' with the background-subtracted OD 590 for the unknowns.<br>Protein concentration of the unknown = x μg/ '''5.0 μL'''.
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</div>
</div>

Current revision

<- Back to Protocols

ELISA Assay
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.

MATERIALS

  • 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

PROCEDURE

Cell harvesting and counting

  1. Harvest cells following the standard procedure.
  2. Resuspend the pelleted cells in 1 mL FACS buffer (1% FBS in 1xPBS) and place them on ice.
  3. Filter 750 uL of the cells through a strainer cap.
  4. Transfer 250 uL of cells to a 1.5 mL microfuge tube. Keep on ice.
  5. Use the remaining 500 uL cells for cell counting by flow cytometry. Set the sampling volume to 20 uL. Calculate cells per uL.

Cell lysis

  1. Transfer 50,000 cells to a 1.5 mL microfuge tube.
  2. Standard protein prep procedure.

Bradford assay

  1. Perform a Bradford Assay to calculate the concentration of total protein in your samples.
  2. Store the samples at -20°C or continue to the next step.

Protein-well binding

  1. Make 1x femto-TBST: Dilute the 10x femto-TBST to 1x in molecular biology-grade water. Make 3.5 mL per sample.
  2. Wash the wells: Empty the storage buffer from the wells. Wash the wells three times with 200 uL 1x femto-TBST
  3. Dilute the protein samples to a final concentration of 1 - 20 ug/mL in 1xPBS, final volume of 100 uL.
  4. Transfer the 100 uL diluted protein samples to the Amine Binding wells.
  5. Incubate at room temperature for 1 hour.
  6. Empty the liquid into a waste container. Eliminate residual liquid by tapping the inverted tubes on a paper towel.

Primary antibody

  1. Make 1x NAP-Blocker: Dilute the 2x NAP-Blocker to 1x in 1x femto-TBST. Make 600 uL per sample.
  2. 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.
  3. Dilute the primary antibody to a suitable concentration in 100 uL 1x NAP-Blocker (per sample). Add 100 uL diluted antibody to each well. Empty the liquid and gently tap out residual liquid onto a paper towel.
  4. 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.

Secondary antibody-HRP

  1. Add 100μl HRP‐labeled secondary antibody solution (diluted in 1x NAP‐Blocker) to each well and incubate for 1 hour at room temperature. After incubation, empty the plate and gently tap out the residual liquid.
  2. 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.
  3. 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.

Signal detection

  1. 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

REFERENCES

  1. G Biosciences. Well-Coated Amine Bonding 96-well plates. http://www.gbiosciences.com/PDF/Protocol/Well-Coated_Amine_Binding_Plates.pdf
  2. G Biosciences. femtoELISA-HRP Manual. http://www.gbiosciences.com/PDF/Protocol/femtoELISA-_HRP_Kit.pdf
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