Antibodies against phosphopeptides recognize proteins that are only in the phosphorylated state. The antibody does not cross-react with unphosphorylated proteins of the same species or other phosphoproteins. Since the phosphorylation state of a protein usually reflects the functional state or activity of a protein, the use of such antibodies is a convenient means of detecting the functional state of a protein.
Author: J.E. Colligan et al, Translated by Xuitao Cao et al. This experiment is from "Comprehensive Immunology Laboratory Guide".
Operation method
Preparation of antibodies recognizing specific tyrosine phosphorylated polypeptides Move Basic Scheme 1 Preparation of polyclonal anti-phosphopeptide antibodies Materials for the preparation of polyclonal anti-phosphopeptide antibodies B S A - Agarose Affinity Substrate (Sigma) packed (as in Supporting Solution 2) on a chromatography column with a bed volume of IOml. Phosphorylated tyrosine affinity matrix columns (IOml bed volume; see Supporting Scheme 3) Crude serum from rabbits immunized with phosphopeptide-B S A couplings PB S / sodium azide. -PBS (Appendix 1) containing 0.02% (m/V) sodium azide (can be stored at 4°C or room temperature for long periods of time) 3m ol/L NaSCN Homogeneous non-phosphopeptide affinity matrix column (3 ml column bed volume; see Supporting Solutions 1 and 2) Homologous phosphopeptide affinity matrix columns (optional; 3 ml bed volume; see Supporting Solutions 1 and 2) Positively selected phosphopeptide affinity matrix columns (3 ml bed volume; see Supporting Solutions 1 and 2) 3.5m ol/L and 4. 5m ol/L MgCl2 (optional) Analyzer bag (MWCO 12,000 to 14,000; 10m m wide, 6.4m m diameter; e.g. Spectrum's Spectra/Por 4) Note: All of the following chromatographic steps are performed at room temperature and the liquid is loaded onto the column by gravity. 1 . Attach the washed B S A -agarose to the phosphotyrosine affinity matrix chromatography column. 2 . Flow 15 m l of crude serum by gravity through both columns, washing with PBS/sodium azide until all yellow serum has passed through the column (or measure the A 28 absorbance of the effluent spectrophotometrically until baseline is reached). Wash the column again with 5-IO ml of PBS/sodium azide and collect all effluent, which may contain the target antibody. The volume of the serum increases with each pass of the column, which prolongs the time to the next pass of the column. If the progress of fluid flow is determined by observing the degree of yellowing of the serum, the more diluted the serum is, the less visible it will be. 3. Optional: A portion of the crude serum fraction and the first pass-through are retained for subsequent analysis and comparison of each purified fraction. If necessary, samples containing multiple phosphorylated tyrosine proteins can be analyzed immediately by immunoblotting (Unit 12. 5) to exclude non-reactive fractions. Alternatively, partially purified fractions may be retained for subsequent analyses and then continued to the next step. 4 . After the serum has been passed through the column, regenerate the column with 10 times the bed volume of 3 mol/L N a S C N and 10 times the bed volume of P B S / sodium azide, and store in P B S / sodium azide at 4°C for backup. 5 . Pass the dressed serum fractions through the same non-phosphopeptide affinity matrix column as many times as possible to minimize cross-reactivity. Regenerate the column with 10 times the column bed volume of 3 mol/L N a S C N and 10 times the column bed volume of PBS/sodium azide. Analyze serum from each or multiple column passes or retain some fractions for later analysis. 6 . If cross-reactivity with a homologous phosphoprotein is expected, pass the dressed serum fraction through the homologous phosphopeptide affinity matrix column using the method provided in Step 5. 7 . Prior to collecting the positively selected affinity-purified breakthrough peak, hydrate and wash a 25 cc m dialysis bag. One end of the bag is clamped with a dialysis clamp, checked for leaks, and 6L of PBS/azide is prepared and stored at 4°C for use as dialysate. 8 . Pass the flow-through serum from the previous chromatography step through a positively selected phosphopeptide affinity matrix column 3 times (to maximize antibody-matrix interaction), without washing the column between each pass. 9 . Collect the last flow-through serum, wash the column with 5 to 20 m l of PBS/sodium azide (depending on pre-column volume and column bed volume), combine the washings with the flow-through serum, and wash the column with an additional 20 m l of PBS/sodium azide, collecting the flow-through liquid as the "wash" peak . 10. Elute with a variety of promoter solvents: either 20m l of 3mol/L NaSCN (optimal) or 10m l of 3.5mol/L MgCl2 (may produce a precipitate, slow the flow rate) followed by IO m U.5mol/L MgCl2. When elution is initiated, immediately collect 3m l of each eluate into the dialysis bag prepared in step 7, clamp the proximal bag opening with dialysis clips and immediately drop into PBS/sodium azide dialysate. Alternatively, 3m l of eluate can be collected into the dialysis tubing and the dialysate added to the tubing as soon as collection of each component is complete. Collect at least 6 fractions (most antibodies in the first 3 fractions, 2 to 3 column bed volumes) and regenerate the column as described previously (step 4). 11. Thoroughly dialyze all fractions collected in Step 10 in PBS/sodium azide at 4°C. 12. Determine the protein concentration of the dialyzed fractions by measuring the absorbance at 280 nm or by protein chromatography and calculate the yield (15 ml of serum to > l m g of purified antibody). Store dispensed antibodies at 70°C and store antibodies in use at 4°C. 13. Analyze the reactivity and cross-reactivity of the final samples as well as the purified fractions stored in the previous steps by ELISA (Unit LI) or immunoimprint (Unit 12.5). Candidate hybridoma cell lines after fusion (Unit 1.3) D M E M /H T Complete Medium Screening dilutions Homophilic phosphopeptide-B S A coupling (as ELS A antigen; Supporting Program 1 and Module 13.1, Module 13.3) Negative Control: Pre-immunization serum for the preparation of hybridoma cell line mice Positive control: used to prepare immune serum for hybridoma cell line mice. homotypic non-phosphotyrosine peptide-BSA coupling (as ELSA antigen; Unit 13.1, Unit 13.3) Non-homologous phosphotyrosyl peptide-BSA coupling (as an ELISA antigen; 13.1, 13.3) Homologous phosphopeptide coupled to BSA (optional; acts as an ELISA antigen; UNIT 13.1, UNIT 13.3) 9 6-well polystyrene tissue culture plates Grid Recording Paper 1 . Fused candidate hybridoma cell lines are inoculated at low density (aiming for 1 cell per 3 wells) in 96-well polystyrene tissue culture plates (Unit 1.3) with HT medium and assayed at approximately 2 weeks. All monoclonal hybridoma culture wells are identified as possible candidates by recording the number of clones in each well on 96 grid recorders, and each well containing a single clone is labeled below the plate to facilitate identification. To facilitate identification, each well containing a single clone was labeled on the underside of the well. 2 . Using aseptic technique, pipette an aliquot of supernatant (e.g., IOOm I from 20,004) from the wells of each possible candidate strain into another 96-well plate (screening plate), recording the number and location of the wells of the original plate, as well as the number and location of the wells of the corresponding plate, on gridded recording paper. The original wells are supplemented with 100 ul of fresh H T medium pre-warmed at 37°C per well. If desired, label the surface of the plate cover with a brightly colored, well-sized adhesive sticker. If the supernatant is not immediately tested, store the screening plate in a plastic bag at 4°C . 3 . Add 150ul of Screening Diluent per well of supernatant in the screening plate (to prevent microbial contamination and to enlarge the volume). 4 . Perform an ELISA (Unit 1.1) using the homologous non-phosphopeptide-B S A coupler as the antigen on a portion of the supernatant from each candidate hybridoma in the screening plate (e.g., 50M1 ). The number and location of wells in the screening plate and the corresponding original plate number and location of wells were recorded for each positive sample. Pre-immunization serum from the mouse used for fusion (negative control) and immunization serum from the same mouse (positive control) were also determined, and were diluted at 1 : 1,000 in the H T medium : screening diluent 1 : 1 mix. The blank screening dilutions were also tested for E L I S A (as an additional negative control). 6 . Expand the clone into culture for all tests required for screening. Freeze some of the fractions and set aside. Subclone the remaining cells by finite dilution (unit 1 . 3 ) to subclone the remaining cells. 7 . As in steps 4 and 5, the supernatants of the subclones are screened for sustained antibody secretion and specificity. Distinguish representative independent subclones from their original parent clones because it is difficult to predict whether each parent monoclonal antibody recognizes the same phosphorylated whole protein or whether it can be used for various types of immunodetection later. Extended culture and freezing of candidate subclones. It is desirable to test for antibody specificity and persistence of secretion serially after multiple rounds of passaging and freezing, recovery, and re-expansion of the culture (recommended). 8 . Subclones are further characterized, the most useful clones are selected, and the culture supernatant is analyzed for reactivity with homophosphorylated whole proteins in specific immunodetection assays (e.g., immunoprecipitation, as described in Monograph 12. 2; or immunoblotting, as described in Monograph 12. 5). 9 . Optionally, antibodies may be prepared by mass culture of the target clone to obtain culture supernatant and affinity purification (see Basic Scheme 1), or ascites may be prepared (Unit 1.4), which may be precipitated by ammonium sulphate and then affinity purified. If necessary, subtyping can be performed using commercially available kits (e.g., Pierce or Sigma). Currently, most peptides containing phosphotyrosine are synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) phosphotyrosine. Due to the lack of a side-chain protecting group on the phosphotyrosine (Kitas^ d . 1994), the standard F m o c synthesis and cleavage steps were applied (Chang and Meienhofer, 1978); however, to achieve efficient coupling of amino acids, higher superconcentrations of activated F m o c amino acids than those used in the standard steps were required after addition of unprotected phosphotyrosine. In some cases, a double coupling method is required, in which the addition of tyrosine phosphate is followed by the addition of small amounts of other amino acids to extend the peptide chain. This alternative method requires the sequential and repeated addition of the next specified amino acid as required to synthesize the peptide. Methods for coupling peptides to affinity matrices are described in this unit (see Supporting Option 2), and common methods for coupling peptides to carrier proteins are described in Units 13.1 and 13.3. This protocol describes the coupling of phosphopeptides and non-phosphopeptides to Affi-Gel 1 0 affinity matrices for the affinity column chromatographic purification of antibodies. This step generates a 3 m l final column bed volume of affinity resin with 3 umol of peptide coupled per m l of gel . Synthetic oligopeptides for coupling (see Supporting Scheme 1) Dimethyl sulfoxide (D M S O ) iV-methyl morpholine (99% pure; AcrosOrganics) Affi-Gd 10 (Bio-Rad) or similar activation support substrate Aminoethanol 0.lmol/L amino ethanol -H C 1, p H 8. 0 High salt/high p H solution - 0 -5mol/L NaCl/0. 4 % (m A O sodium bicarbonate) High salt/low pH solution: 0.5 mol/L NaCl/100 mmol/L sodium acetate, pH 4.2 PBS/Sodium azide: PBS containing 0.02% (m/V) sodium azide (Appendix 1). 0.5mol/L NaCl 3mol/L NaSCN Polypropylene screw cap centrifuge tube End-over-end shakers Vacuum Attractor Glass chromatography column, > 5 ml capacity (Bio-Rad), or 5 ml plastic syringe with 1-cc siliconized glass wool stopper 1 . Dissolve the synthesized oligopeptides in DMSO at a volume ratio of 0.5 to 4 times the desired column volume. For example, a 9 mmol peptide (15.3 mg, for a peptide with an average molecular mass of 15 mEr and 1700 molecules) should be dissolved in a 3 ml bed volume. 2 . Neutralize the peptide solution by careful titration as follows: add the peptide synthesis grade N-methyl-p-oxoazacycline (at high concentration or diluted to 1 : 1 ~ 1 : 9 in DMSO) in 1 to 2ul increments, remove a 2 to 3ul aliquot after each addition, dilute it with 50ul of water, and spot it on a p H test strip. Repeat this step until the p H reaches 7 to 8 (basically 7 to 8 ul per 3 umol of peptide, depending on the amino acid sequence). 3 . Mix the contents of the Affi-Gd 10 Affinity Matrix vial well to suspend the resin, then transfer the desired volume of resin [2 times the column volume (4 times the 50 % suspension)] to a polypropylene screw-cap centrifuge tube and wash three times in DMSO, each time centrifuging the vial for 5 min at room temperature at 700 g. Aspirate the supernatant, resuspend the resin in 5 times the volume of DMSO, and centrifuge the vial again at 700 g. Centrifuge again at 700 g. Do not exceed the recommended speed. Do not exceed the recommended speed or the resin may be damaged. 4 . Aspirate excess D M S O from the resin, add to the peptide solution prepared in Step 2, and incubate overnight at room temperature with an upright cylinder shaker. Add pure aminoethyl alcohol (to eliminate unreacted ester groups) per milliliter of resin and incubate for 2 h at room temperature with an upright cylinder shaker, washing twice in D M S O as in Step 3. 5 . Wash twice in 0.lmol/L aminoethanol-H C l at p H 8.0 as in step 3 (the first wash should be done on ice because of the large amount of heat generated), remove O.lmol/L aminoethanol ^ H C l after the second wash, replace with a new solution, and incubate overnight at 4°C on an upright cylinder shaker before centrifuging at low speed and aspirating the supernatant. 6 . Using the method described in step 3, the resin is washed three times with high salt/high p H solution, three times with high salt/low p H solution, and three times with P BS/sodium azide, and the washed resin is stored at 4°C until mounted on the chromatography column. 7 . The matrix is mixed into a suspension with O.5 mol/L NaCl and poured into the bottom of a glass chromatography column or a 5-ml plastic syringe with a 1-cc siliconized glass wool stopper, and each column is washed with 10 times the volume of the column with 3 m d/L NaCl and 10 times the volume of the column with PBS/sodium azide. This step generates an IOml final column bed volume of affinity resin with 3 umol of tyrosine phosphate coupled to Affi-Gd 1 0 per milliliter. Tyrosine phosphate 0.4% (m / V ) sodium bicarbonate lmol/L N a O H (optional) Aminoethanol 0 -lmol/L amino ethanol -H C 1, p H 8. O Sintered glass funnel and vacuum aspirator Glass chromatography columns, capacity > 1 4 ml (Bio-Rad) 1 . Dissolve tyrosine phosphate at a volume ratio of 0.5 to 4 times the expected column bed volume in 0 . 4 % sodium bicarbonate. For example, 30 umol of phosphotyrosine (7.8 mg, for a relative molecular mass of 261 phosphotyrosine) corresponds to a column bed volume of IOml. For simplicity, more tyrosine phosphate can be used because it is relatively inexpensive. 2 . Determine p H with a p H test strip to 7 to 8. If adjustment is necessary, neutralize the solution by titration, e.g., by adding a small amount of l m d /L N a O H , removing 2 to 3 jlJ aliquots with each addition, and then dotting on the p H test strip. Repeat this procedure until the p H reaches 7 to 8. 3 . Mix the contents of the Affi-Gel 10 Affinity Matrix vial well to suspend the resin, then transfer the desired volume of resin [2 times the column volume (4 times the 50 % suspension) of resin] to a glass-ceramic funnel attached to a vacuum aspirator. Wash 3 times, each time filling the resin with cold distilled water and suctioning, and then wash in the same way with ice-cold 0.4 % sodium bicarbonate. 4 % sodium bicarbonate. 4 . Remove most of the excess liquid from the gel by vacuum aspiration without completely drying it, transfer it to a screw-cap centrifuge tube, add the tyrosine phosphate solution prepared in step 2, and do not allow more than 20 min between the removal of the resin from the bottle (step 3) and mixing it with the tyrosine phosphate solution. incubate the gel overnight at 4°C on an upright cylindrical shaker. 5 . Add 2ul of pure aminoethyl alcohol per ml of resin (to eliminate unreacted ester groups) and incubate for 2 h at room temperature with an upright cylinder shaker. Wash the resin twice, each time by low speed centrifugation (see Additional protocol 2, step 3) and aspirate the supernatant, and resuspend the resin in 5 times the volume of 0.4 % sodium bicarbonate. The resin was resuspended with 5 times the volume of 0.4 % sodium bicarbonate and centrifuged again at low speed. 6 . Wash the resin twice in 0.lmol/L ethylamine-HCl at p H 8.0 as in step 4. After the second wash, remove the 0.lmol/L ethylamine-HCl and replace it with a new solution. After the second wash, remove the 0.lmol/L aminoethyl alcohol-HCl and replace it with a new solution. Incubate overnight at 4°C in an upright cylinder shaker, then centrifuge at low speed and aspirate the supernatant. Wash, store, and fill the column with resin (see Option 2, Steps). For more product details, please visit Aladdin Scientific website.
The
5 . ELISA (Unit 1 . 1 ) of a portion of the fraction of each positive sample screened in Step 4, either in sequence or simultaneously, using the homozygous nonphosphorylatable peptide-B S A coupling as the antigen; non-homozygous irrelevant phosphorylated reactive clones are rejected by the ELISA (Unit 1 . 1 ) test to reject non-phosphorylated reactive clones, and an E L IS A test with a non-homologous unrelated phosphotyrosine peptide-B S A coupler as the antigen to reject clones with other phosphotyrosine reactivities. In addition, if possible cross-reactivity with homologous phosphorylated proteins is anticipated, the BSA-coupled homologous phosphopeptide is used as the antigen to weed out any possible homologous phosphorylated protein cross-reactivity. If necessary, clones with specific reactivity to non-phosphorylated cognate peptides can also be screened at the same time.
Wash, store and fill the column with resin (see Option 2, step 7).