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

The overall goal of this procedure is to increase specificity of antibodies against closely related proteins by removing cross-reactive antibodies. This is accomplished by first identifying protein domains of greatest divergence between homologous proteins. The second step is to express and purify these protein domains.

Next, these protein domains are coupled via the N or C terminal to a chromatography matrix. Spheros is used in this demonstration. The final step consists of incubating immune serum with resins coupled to each cross-reactive domain.

Ultimately, Eliza and Western blotting are performed to assess the efficacy of the procedure. The main advantage of this technique over existing methods like the use of monoclonal antibodies or affinity purification, is that we can maintain affinity against the desired target while eliminating known cross-reactive epitopes. Generally, individuals new to this method will struggle because the protein they're trying to study is unstable.

Demonstrating this procedure will be Dr.Uri Kim, a postdoctoral fellow from our laboratory. This study requires two columns, one for neuron derived orphan receptor, one or nor one and the other for NER growth factor IB or nor 77. Since the same protocol applies to both proteins, only the preparation of one column will be demonstrated.

Thaw the protein to be coupled to the resin on ice, and then determine the protein concentration using its molar extinction coefficient and its absorbance at 280 nanometers. Use two milliliters of resin for two milligrams of protein. All steps that follow are for two milliliters of resin.

In a 10 milliliter column, prepare a 10 milliliter column by pushing a FR to the bottom and running PBS pH 7.4 through it. Cap the bottom of the column and add two milliliters of coupling buffer. Add four milliliters of the slurry to the column to obtain two milliliters of resin.

Remove the bottom cap and let the column drain. Rinse the column with a total of six milliliters or three resin bed volumes of coupling buffer and allow the column to drain. After the coupling buffer is drained, replace the bottom cap.

Next, add two to four milliliters of protein suspended in coupling buffer to the column. Keep an aliquot of the protein solution to assess the coupling efficiency. Later cap the column and mix end over end.

To make a homogenous solution, measure the reactions total volume, taking the resin volume into consideration and add 10 microliters of five molar sodium sano boro hydride per milliliter of reaction. Cap the column and mix End over.End. Secure the column on a tube rotator and let the reaction continue overnight.

At four degrees Celsius on the following morning, take the column to the chemical hood carefully remove the cap as some gas may have formed. Drain the column into a clean tube and save the ilu. The protein concentration of the IIT will be measured and compared with the starting protein concentration.

To assess the coupling efficiency, wash the resin with four milliliters of coupling buffer and let the column drain. Once the ligand binding domain or LBD has been coupled to the resin, the next step is to block the remaining sites. To start this procedure, wash the resin with four milliliters of quenching buffer drain and replace the bottom cap.

Add two milliliters of quenching buffer and suspend the resin. Assess the total reaction volume by measuring the resin volume and the quenching buffer volume. Add 10 microliters of five molar sodium cyana boro hydride per milliliter of reaction volume.

Mix gently at room temperature for 30 minutes. End over end After 30 minutes, take the column to the fume hood. Carefully remove the top and then remove the bottom cap and let the column drain into a waste tube.

Wash the column with five resin volumes of wash solution. Monitor the washes for the presence of residual proteins by measuring the ouit absorbance At 280 nanometers uncoupled proteins are washed out by the high salt concentration. Lastly, wash the resin with six milliliters of DGAs PBS containing 0.02%Sodium azide, keep the column at four degrees Celsius until needed.

Begin the procedure for purifying nore one specific antibodies by rinsing the NORE 77 LBD coupled column with 10 milliliters of PBS and letting the column drain cap the bottom of the column and place the drained neuro 77 LBD coupled column in a new 15 milliliter collection tube. Add five milliliters of protein, a purified anti neuro one antibodies to the column and cap it. Place the column on a rotator at four degrees celsius for one hour.

Next, remove the top and bottom caps and collect the flow through. Set the flow through aside on ice until it is time to add it to the nor. One LBD coupled column.

Rinse the no one LBD coupled column with 10 milliliters of PBS and let the column drain cap the bottom of the column and place the nor one LBD coupled column in a 15 milliliter collection tube. Add the flow through from the north 77 LBD coupled column. Cap the column and place on a rotor at four degrees Celsius for one hour.

After one hour. Remove the top and bottom caps and collect the flow through. Measure the antibody concentration by measuring the absorbance at 280 nanometers.

The purified anti nore one antibody is subsequently analyzed by Eliza. Add 100 microliters of protein to the wells of a 96 well plate. If testing, three different proteins add 100 microliters of protein one to wells, a one to H four, 100 microliters of protein, two to wells, a five to H eight, and 100 microliters of protein three to wells a nine to H 12.

Cover the plate and incubate overnight at four degrees Celsius on the following morning. Wash the coated wells twice with 300 microliters per well of PBS. Then add 300 microliters of Eliza blocking buffer to all the antigen coated wells and incubate at room temperature for two hours.

After two hours of blocking, replace the ELIZA blocking buffer in each well with 100 microliters of fresh Eliza blocking buffer. Add 100 microliters of diluted purified anti-EU one antibody to all the wells in row. A incubate at room temperature for one hour after one hour.

Wash the plate three times with 0.05%tween 20 after the third wash, blot the plate to remove excess wash buffer. Add 100 microliters of horse radish peroxidase, conjugated goat anti rabid IgG diluted in Eliza blocking buffer to each well incubate at room temperature for one hour after one hour. Wash the plate three times with 0.05%tween 20 as shown earlier after the third wash.

Rinse all wells by filling them with 300 microliters of deionized water, blot excess water by inverting the plate onto absorbing paper. Add 100 microliters of three three prime, five, five prime tetraethyl benadine, warm to room temperature to all wells and incubate for 15 to 30 minutes in the dark at room temperature. Stop the reaction by adding 50 microliters of two normal S uric acid to each well.

Lastly, read the absorbance at 450 nanometers. Subtracting the background at 650 nanometers A comparison of protein. A purified no one specific antibodies with protein.

A purified anti Noro one antibodies followed by passage through no 77 LBD and no one LBD columns shows that while protein a purified anti no one exhibited strong binding to no one LBD. It also showed significant binding to no 77 LBD and to no one LBD Further purification of anti no one against no 77 LBD and no one LBD completely removed this cross reactivity. This specificity was further demonstrated by Western blot analysis of extracts from cells transfected with expression vectors carrying Mick tagged full length nor one nor one and nor 77 anti mic antibodies revealed that each full length protein was expressed at its expected molecular weight protein, a purified anti nor one robustly detected full length nor one, but also detected nor one and nor 77 although less efficiently.

In contrast, the fully purified nor one antibody did not exhibit any detectable cross reactivity to nor one and nor 77 immunohistochemical analysis of midbrain dopamine neurons with the fully purified nor one antibody confirmed the prominent expression of nor one immunohistochemistry Using tyrosine hydroxylase further revealed that neuro one is also exclusively expressed in the nucleus of mid-brain dopamine neurons in the substantial nigra area Once mastered this technique can be done in two days if it is performed properly. Don't forget that working with sodium ion or hydride can be extremely hazardous and precautions such as working in a chemical hood should always be taken while performing this procedure.