Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs, Nor1 and Nur77

Summary

Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.

Abstract

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson’s disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.

Introduction

The transcription factor Nurr1 and its homologs (Nur77 and Nor1) belong to the nuclear receptor subfamily 4A (NR4A)¹. They are also orphan receptors because their endogenous ligands are not identified yet. Nurr1 was first cloned in 1992 and although known to be expressed in the brain², its essential role for development and maintenance of midbrain dopamine neurons was revealed by knockout mouse studies³. In addition, its important role for maintenance of midbrain dopamine neurons was recently demonstrated by a conditional knockout study⁴. Due to these elegant studies showing Nurr1’s critical roles for midbrain dopamine neurons, many subsequent studies have largely focused on the midbrain dopamine neurons and dopamine-related neurodegenerative disorder, Parkinson’s disease (PD)⁵.

Notably, Nurr1 is expressed not only in the midbrain dopamine (mDA) neurons, but also in diverse brain areas², suggesting that it may have functional roles in many non-DA areas, which is strongly supported by more recent studies showing that Nurr1 plays important roles in various brain functions. For examples, it was shown that memory-inducing activities such as learning, or other hippocampus-dependent tasks result in up-regulation of Nurr1 expression in the hippocampus^6,7. In addition, knock down of Nurr1 expression in the hippocampus was sufficient to impair long-term memory and/or synaptic plasticity^8-11, strongly suggesting that Nurr1 plays diverse roles in many brain areas. Thus, to further understand the cell-type-specific and subcellular expression of Nurr1, it is desirable to use Nurr1-specific antibodies, which do not exhibit any cross-reactivity to its homologs Nur77 or Nor1. This paper describes a pre-adsorption protocol to generate Nurr1-specific antibodies and present additional data showing its specificity.

Protocol

Note: The composition of all solutions cited below can be found in the Materials/Equipment Table.

1. Protein A Column Antibody Purification

1. Equilibrate a protein A spin column and all required buffers to room temperature for 15 min prior to starting the procedure.
2. Dilute the immune serum 1:1 with Protein A IgG Binding Buffer. (5 ml of serum is recommended).
3. Gently tap a protein A spin column on the bench top to dislodge any resin that may be lodged in the cap. Remove the top cap and gently snap off the bottom closure. Place the column in a 15 ml collection tube and allow storage solution to drain.
4. Add 5 ml of Protein A IgG Binding Buffer and allow the solution to drain.
5. Apply the diluted immune serum to the column and collect the flow-through. For best results, add a sample volume that contains a concentration of antibodies less than 80% of the column’s antibody-binding capacity.
6. Wash the column with 15 ml of Protein A IgG Binding Buffer or until the absorbance at 280 nm is below 0.1.
7. Add 100 µl of Neutralization Buffer to five labeled collection tubes.
8. Add 5 ml of IgG Elution Buffer to the Protein A column and collect 1 ml fractions in each of the tubes containing the 100 µl of Neutralization Buffer.
9. Measure the absorbance of each fraction at 280 nm and pool fractions with an absorbance greater than 0.5. Keep the pooled fractions on ice until ready for dialysis.
10. Regenerate the column by adding 8 ml of IgG Elution Buffer and allow the solution to flow through the column.
11. Rinse the column with Protein A IgG Binding solution until the eluent pH returns to 7.4.
12. Store the column by adding 5 ml of storage solution (0.02% sodium azide in PBS). When approximately 3 ml remain in the column, cap bottom and secure the top cap. Store the column at 4 °C.
13. Determine the length of dialysis tubing needed according to the manufacturer’s instructions. Using 16 mm flat diameter tubing use 5.47 cm of tube length per ml of solution to be dialyzed. Cut the tubing and rinse with PBS pH 7.4 for 15 to 30 min.
14. Fold and clip one end of the dialysis tubing, transfer the pooled IgG containing fractions to dialysis tubing equilibrated in PBS pH 7.4 and close the other end.
15. Dialyze at room temperature against 200 volumes of PBS pH 7.4 for 2 hr.
16. Change the dialysis buffer (fresh PBS pH 7.4) and dialyze for another 2 hr at room temperature.
17. Change the dialysis buffer (fresh PBS pH 7.4) and dialyze overnight at 4 °C.
18. Keep the dialyzed antibody solution at 4 °C until ready to proceed with further purification steps.
19. Assess the antibody concentration using its absorbance at 280 nm and the molar absorption coefficient of antibodies at 280 nm of 210,000 M^-1 cm^-112.

2. Coupling of LBD Proteins to AminoLink Coupling Resin

1. Prepare two columns, one for Nor1 and the other for Nur77. Follow the same protocol for both proteins.
2. Thaw the protein to be coupled to the resin on ice. Determine the protein concentration using its molar extinction coefficient and its absorbance at 280 nm¹².
   1. Alternatively, use a protein determination assay such as the bicinchoninic acid assay¹³. If the protein is dissolved in an amine-containing buffer dialyze or buffer exchange against the Coupling Buffer. If the protein is in a suitable buffer (no free amines) dilute it 4-fold in Coupling Buffer.
3. Use 2 ml of resin for 2 mg of protein. All steps that follow are for 2 ml resin in a 10 ml column. Up to 10 mg of protein can be linked per 1 ml of resin (2 ml of 1:1 slurry).
4. Prepare a 10 ml column by pushing a frit to the bottom and running PBS pH 7.4 through it. Cap the bottom of the column and add 2 ml of Coupling Buffer.
5. Add the desired volume of slurry (4 ml to obtain 2 ml of resin) to the column, remove the bottom cap and let the column drain. Rinse the column with a total of 6 ml (3 resin-bed volume) of Coupling Buffer and allow the column to drain. After the Coupling buffer has drained, replace the bottom cap.
6. Add 2-4 ml of protein suspended in Coupling Buffer to the column (keep an aliquot of the protein solution to assess the coupling efficiency by comparing the protein concentration of the eluate in step 2.11 using either the absorbance at 280 nm or the bicinchoninic assay). Cap and mix end over end to have a homogeneous solution.
7. Weigh an empty 1.8 ml microcentrifuge tube and move to the fume hood. Carefully transfer NaCNBH₃ (about 0.05 g) in the pre-weighed tube. Close the tube and weigh the tube containing NaCNBH₃. Do not open the tube outside the hood.
8. Based on the weight of NaCNBH₃ transferred into the tube, make a 5 M solution by adding 1 M NaOH. The molecular weight of NaCNBH₃ is 62.84 g therefore 0.05 g is equal to (0.05/62.84 = 7.96 x 10^-4 moles) 7.96 x 10^-4 moles. To make a 5 M solution add (7.96 x 10^-4 /5) 159 µl of 1 M NaOH.
9. Measure the reaction’s total volume taking the resin volume into consideration. Add 10 µl of 5 M NaCNBH₃ per ml of reaction.
   1. For example: for a 4 ml reaction volume add 40 µl of 5 M NaCNBH₃. For 1 ml of resin and 3 ml of added protein, the total volume is 4 ml.
10. Cap the column and mix end over end. Secure the column on a tube rotator and let the reaction continue overnight at 4 °C.
11. In the morning, bring the column to the chemical hood and carefully remove the cap as some gas may have formed. Drain the column into a clean tube and save the eluate to measure the protein concentration using the absorbance at 280 nm method¹² or the bicinchoninic acid assay and compare it with the starting protein concentration in step 2.6.
12. Wash the resin with 4 ml of Coupling Buffer and let the column drain.

3. Blocking Remaining Sites

1. Wash the resin with 4 ml of quenching buffer. Drain and replace the bottom cap. Add 2 ml of quenching buffer and suspend the resin.
2. Assess the total reaction volume by measuring the resin volume and the quenching buffer volume then add 10 µl of 5 M NaCNBH₃ per ml of reaction volume.
3. Mix gently at room temperature for 30 min end-over-end. After 30 min, bring the column in the fume hood. Carefully remove the top and then remove the bottom cap and let the column drain into a waste tube.
4. Wash the column with 5 resin volumes of Wash solution. Monitor the washes for the presence of residual proteins by measuring the eluate’s absorbance at 280 nm. Uncoupled proteins are washed out by the high salt concentration.
5. Wash the resin with 6 ml of degassed PBS pH 7.4 containing 0.02% sodium azide. Keep the column at 4 °C until needed.

4. Purification of Nurr1 Specific Antibodies

1. Rinse the Nur77 LBD coupled column with 10 ml of PBS pH 7.4 and let the column drain. Cap the bottom of the column and place the drained Nur77 LBD coupled column in a new 15 ml collection tube.
2. Add 5 ml of protein A purified anti-Nurr1 antibodies, cap the column and place on a rotator at 4 °C for 1 hr. Remove the top and bottom caps and collect the flow through. Set aside on ice until ready to add to the Nor1 LBD coupled column.
3. Rinse the Nor1 LBD coupled column with 10 ml of PBS pH 7.4 and let the column drain. Cap the bottom of the column and place the Nor1 LBD coupled column in a 15 ml collection tube.
4. Add the flow through from the Nur77 LBD coupled column, cap the column and place on a rotator at 4 °C for 1 hour. Remove the top and bottom caps and collect the flow through.
5. Measure the antibody concentration by measuring the absorbance at 280 nm.

5. ELISA-based Analysis of Purified Anti-Nurr1 Antibody

1. Add 100 µl of protein (2.5 µg/ml) to the wells of a 96 well plate. If testing 3 different proteins, add 100 µl of protein 1 to wells A1 to H4, 100 µl of protein 2, to wells A5 to H8 and 100 µl of protein 3 to wells A9 to H12. Cover the plate and incubate overnight at 4 °C.
2. Wash the coated wells twice with 300 µl per well of PBS pH 7.4 and add 300 µl of ELISA blocking buffer to the entire antigen coated wells and incubate 2 hours at room temperature.
3. While the plate is blocking prepare a desired starting dilution of purified anti-Nurr1 antibody (ranging from 10 to 100 µg/ml) in ELISA blocking buffer.
4. After 2 hr of blocking, replace the ELISA block buffer with 100 µl of fresh ELISA block buffer to all wells.
5. Add 100 µl of diluted purified anti-Nurr1 antibody to all the wells in row A. Serially dilute the antibodies by transferring 100 µl of solution from row A to the wells in row B followed by transferring 100 µl of solution from row B to row C continuing down to row G. After mixing the solutions in wells in row G discard the extra 100 μl. Wells in row H only receive the initial 100 µl of blocking buffer. Incubate at room temperature for 1 hr.
6. Wash the plate three (3) times with 300 µl per well of 0.05% Tween 20 in PBS pH 7.4 and blot the plate to remove excess wash buffer.
7. Add 100 µl of Horse Radish Peroxidase (HRP) conjugated Goat anti-rabbit IgG diluted in ELISA block buffer (1:8,000 dilution; the typical range is from 1:5,000 to 1:25,000). Incubate at room temperature for 1 hr.
8. Wash the plate three (3) times as described in 5.6. Rinse all wells by filling them with 300 μl of deionized water. Blot excess water by inverting the plate onto absorbing paper.
9. Add 100 µl of 3, 3’, 5, 5’ tetramethylbenzidine (warmed to room temperature) to all wells and incubate 15 to 30 min in the dark at room temperature.
10. Stop the reaction by adding 50 µl of 2 N H₂SO₄.
11. Read absorbance at 450 nm subtracting background at 650 nm.

Results

A comparison of Protein A column purified Nurr1-specific antibodies with Protein A purified anti-Nurr1 antibodies followed by passage through Nur77 LBD and Nor1 LBD columns is shown in Figure 1. As can be seen, Protein A-purified Nurr1 antibody exhibited a strong binding to Nurr1 LBD. However, it also showed significant binding to Nur77 LBD and to Nor1 LBD. When Protein A-purified Nurr1 antibodies were further purified against Nur77 LBD and Nor1 LBD, the final affinity purified Nurr1 antibodies exhibited...

Discussion

The success of this protocol relies on the availability of pure proteins for characterization of the antibodies raised against a specific member of the protein family of interest. There is no need to purify the antibodies using protein A/G until it is clearly established that there are significant cross-reactivities by ELISA and Western blotting.

As stated in the text, it is important to avoid suspending the protein(s) to be cross-linked to the column in an amine-containing buffer such as Tris...

Materials

Name	Company	Catalog Number	Comments
Purified Ligand Binding Domain from Nurr1, Nor 1 and Nur77			Column Storage solution
AminoLink Coupling Resin and Kit	Thermo Scientific	44890
Protein A spin column	Thermo Scientific	89978
Dulbecco's Phosphate-Buffered Saline	Corning	21-031-CV
96 well Flate-bottom plates, High Flange, 330μL	Thermo Scientific	3455
10% Normal Goat Serum	KPL	50-675-69
Milk Diluent/ Blocking Concentrate	KPL	50-82-01
IgG Elution Buffer	Thermo Scientific	21004
UltraPure 1 M Tris-HCI Buffer, pH 7.5	Life Technologies	15567-­‐027
Micro BCA Protein Assay Kit	Thermo Scientific	23235
Horse Radish Peroxidase conjugated Goat anti-Rabbit IgG (H+L)	Thermo Scientific	31460
Ni-NTA Resin	Thermo Scientific	88221
3, 3', 5, 5' Tetramethylbenzydine	Thermo Scientific	34028
2N H2SO4	Macron Fine Chemicals	H381 05
Protein A IgG Binding Buffer	Thermo Scientific	21001
IgG Elution Buffer	Thermo Scientific	21004
Neutralization Buffer			1M Tris-­‐HCl pH 8.5
Column Storage solution			Phosphate Buffered Saline containing 0.02% sodium azide
Spectra/Por 7 pre-treated dialysis tubing	Spectrum Labs	132128
10 ml Disposable columns	Thermo Scientific	29924
AminoLink Coupling Buffer			0.1M sodium phosphate, 0.05% NaN3, pH 7.0
Quenching buffer			1M Tris. HCI, 0.05% NaN3, pH 7.4
Wash Solution			1M NaCl, 0.05% NaN3
ELISA Blocking buffer			1% Normal Horse serum (NHS), 1% Normal Goat Serum (NGS) in 1X KPL milk diluent
Storage Solution			PBS pH 7.4 containing 0.02% sodium azide
Micropipettes: 10 μl; 20 μl; 200 μl; 1000 μl