Guinea pigs were immunized with the KLH-conjugated peptides

Guinea pigs were immunized with the KLH-conjugated peptides. fruA25 relA1 rps150(strR) rbsR22 deoC1]. The protein was purified to electrophoretic homogeneity by chromatography on an Ni-agarose column [16]. Immunization of rabbits was performed with 0.5 mg of the purified protein in 0.5 mL RIBI adjuvant, followed by booster injections with 0.5 and 0.3 mg on days 14 and 21, respectively. The antiserum was collected on day 28. Monospecific antibodies were prepared following the method explained by Olmsted [17]. Briefly, 2 mg of purified LCK (phospho-Ser59) antibody antigen was blotted on nitrocellulose after SDS electrophoresis. The protein band was marked with Ponceau PD 334581 answer and slice out. After blocking of the membrane strip with 1% low-fat milk powder in phosphate-buffered saline, the membrane was incubated with the antiserum for 1 hour, followed by considerable washing with Tris-EDTA-buffered saline. The antibodies were eluted with 0.2 M glycine (pH 2.0) for 2 moments, followed by immediate neutralization with 1 M triethanolamine. The specificity of the PTPIP51 antibody was tested by ELISA and by immunoblotting of the isolated purified recombinant protein staining bands with 52 kDa, 34 kDa, and 30 kDa. Immunoblotting of homogenates from porcine spleen tissue revealed bands of 48 kDa, 40 kDa, and 29 kDa [18]. The antibody binds to the EGFP fusion PTPIP51 protein expressed in HEK293 [19]. Preabsorbing the PTPIP51 antibody against its antigen completely abolished the immune reaction in all tested samples [20,21,22]. 2.4. Peptide Specific Phospho-Tyrosine 176 PTPIP51 Antibody For analysis of the tyrosine phosphorylation state of PTPIP51, an antibody (BioLux, Stuttgart, Germany) to the tyrosine 176 phosphorylated sequence DAESEGGYTTANAE was used (P51ab-PTyr). Identity and purity of the synthetized PD 334581 peptide was approved by ESI-MS and UV-analysis. Guinea pigs were immunized with the KLH-conjugated peptides. The specificity of each antibody was tested by ELISA and Western blot. To verify the use of these peptide specific antibodies for immunostaining, preabsorption experiments were performed. 2.5. Preabsorption Experiments for Immunoblotting Specificity of the PTPIP51 immunoreactivity for both antibodies P51ab and P51ab-PTyr was controlled by preabsorbing both antibodies with the corresponding purified antigen (P51ab: recombinant PTPIP51 full length protein; P51ab-PTyr: phophorylated antigenic peptide explained in Section 2.4) at a concentration of 20 g/mL for 18 hours at 4 C prior to the immunostaining. As positive control, a normal incubation mixture including the same concentration of PTPIP51 antibody was used. Physique 1 displays in the left panel an immunoblot done with the preabsorbed P51ab-antibody. In Physique 1 right panel an immunoblot done with preabsorbed P51ab-PTyr antibody is usually shown. Open in a separate window Physique 1 Control experiments for immunoblotting. First panel: immunoblot done with the PD 334581 preabsorbed P51ab-antibody. Second panel: immunoblot done with preabsorbed P51ab-PTyr antibody. Third panel: Unfavorable control with the omission of the P51ab antibody. Fourth panel: Unfavorable control with the omission of the P51ab-PTyr antibody. 2.6. Immunoblotting Samples of HaCat cell lysate were separated on a 10% SDS-PAGE gel. Transfer on an Immobilon P membrane (Millipore) was performed according to Towbin [23]. The membrane was blocked with 10% fat-free milk powder in PBS. Incubation with polyclonal rabbit anti-PTPIP51 (P51ab) or polyclonal guinea pig anti-pTyr176-PTPIP51 (P51ab-PTyr) was carried out overnight at room heat. Either alkaline phosphatase-conjugated anti-rabbit or alkaline phosphatase-conjugated anti-guinea pig immunoglobulins were applied for 1 h at room heat diluted in 0.5% fat-free milk powder. The reaction was visualized with the SigmaFast BCIP/NBT substrate. A prestained molecular excess weight marker (Biorad, Cat# 161-0374) was utilized for calibration. 2.7. Fluorescence Microscopy The Axioplan 2 fluorescence microscope equipped with Plan-Apochromat objectives.