Validation data of a rabbit antiserum and affinity purified polyclonal antibody against the N-terminus of human GPR30

Rabbit antiserum was generated against the N-terminus of human GPR30 followed by peptide affinity purification. In this article, the methodology used and validation data are presented. The peptide affinity purified polyclonal antibody specifically detects human GPR30 in ELISA and on western blots of total protein prepared from human breast cancer cell lines.


a b s t r a c t
Rabbit antiserum was generated against the N-terminus of human GPR30 followed by peptide affinity purification. In this article, the methodology used and validation data are presented. The peptide affinity purified polyclonal antibody specifically detects human GPR30 in ELISA and on western blots of total protein prepared from human breast cancer cell lines.

Value of the data
The antibody generated can serve as a tool for basic and clinical research in the field of GPR30 biology.

Data
After immunization of two rabbits (A and B) with the peptide antigen, the antiserum harvested from the third bleed of rabbit B that was collected after the seventh booster, was found to be the most reactive compared to pre-immune serum (Fig. 1). Western blots of total protein prepared from human cell lines with this antiserum resulted in the detection of $ 52 kDa band of GPR30 along with other non-specific proteins, which were also detected by pre-immune serum or secondary antibody alone (Figs. 2 and 3). The affinity purified antibody obtained from the antiserum (third bleed) of rabbit B showed similar reactivity to that of the antiserum (Fig. 4). It produced clean western blotting results, in which, only one $ 52 kDa band of GPR30 was detected (Fig. 5).

Experimental design
Rabbits were immunized with N-terminus peptide of human GPR30 and hyperimmune serum was collected after several boosters. Immunoreactivity of the immune serum was checked by indirect ELISA and the antiserum with the highest reactivity was tested for the specificity by western blotting. Upon confirmation of specificity, immunoglobulins were affinity purified and reconfirmed by indirect ELISA and western blotting.

Materials and reagents
Plasticware for cell culture was from Tarsons (Kolkata, India) and Greiner Bio-One (GmbH, Germany). Phenol red-containing media (DMEM and RPMI-1640) and fetal bovine serum (FBS) for cell culture were from Gibco (NY, USA). Radioimmunoprecipitation assay (RIPA) buffer was purchased   5. Detection of GPR30 in total protein by affinity purified antibody. Proteins were fractionated by 10% SDS-PAGE under denaturing conditions and transferred to nitrocellulose membranes. Membranes were subjected to western blotting analysis followed by chemiluminiscence detection. The anti-β-actin antibody was used in a dilution of 1:5000 and the affinity purified primary antibody was used in a dilution of 1:15,000.

Generation of polyclonal antibody and affinity purification
Polyclonal antibody generation and peptide affinity purification was performed at Abgenex Pvt. Ltd., Bhubaneswar, India. N-terminus peptide (MDVTSQARGVGLEMYPGTAQPAA) [1] of GPR30 was chemically synthesized with an extra cysteine residue at the C-terminus of the peptide. The peptide was cross-linked to Keyhole Limpet Haemocyanin (KLH, Pierce, Cat. #77600) using maleimidesulfhydryl chemistry. KLH was activated by treating with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC, Pierce, Cat. #22322). Maleimide-activated KLH was then purified by gel filtration chromatography and mixed with peptide for cross-linking. Efficiency of conjugation was assessed by determining the free sulfhydryl groups before and after conjugation using Ellman's reagent (Pierce, Cat. # 22582). Two rabbits (A and B) were immunized with antigen (KLH conjugated peptide) in Complete Freund's Adjuvant (CFA) or Incomplete Freund's Adjuvant (IFA) after the collection of pre-immune serum. The first immune serum was collected after primary immunization (200 mg antigen/rabbit in CFA) and five boosters (100 mg antigen/rabbit in IFA). Subsequently, the next two batches (second and third bleeds) of immune sera were collected after 6 th and 7 th boosters, respectively. First and third batches of immune sera were tested for immune reactivity by indirect ELISA (Fig. 1). Pre-immune serum served as negative control. Amongst the bleeds collected, the third bleed of rabbit B was found to be the most reactive (Fig. 1). The immune serum from third bleed of rabbit B detected the $ 52 kDa band of GPR30 ( Fig. 2A) which is consistent with other reports [2,3]. Several non-specific bands were also observed. However, the non-specific bands were also detected on western blots probed only with the secondary antibody (Fig. 2B). As shown in Fig. 3A, a specific $ 52 kDa band of GPR30 was detected in breast cancer cell lines along with other nonspecific bands which were also detected by the pre-immune serum (Fig. 3B). This immune serum was affinity purified against the immunogenic peptide using the sulfo-link matrix. Immunoreactivity of the affinity purified antibody was confirmed by indirect ELISA (Fig. 4). This antibody detected a single prominent $52 kDa GPR30 band against a much cleaner background on western blots of total proteins from breast cancer cell lines (Fig. 5).

Indirect ELISA
On day 1, the peptide antigen (200ng/well) was coated on a 96-well plate (Nunc-Immuno plate, Cat. #439454, F96 Cert. MaxiSorp) for 2 h at room temperature followed by overnight incubation at 4°C. On day 2, the plates were kept on a shaker at room temperature for 2 h. The wells were then washed with phosphate buffered saline containing 0.05% Tween 20 (PBST), followed by blocking with 5% skimmed milk in PBST for 1 h. After three washes with PBST, 100 μL per well of primary antibody (diluted 1:5000 in PBST containing 1% skimmed milk) was added to each well and incubated at room temperature. After 2 h the wells were washed as above. 100 μL of HRP-conjugated secondary antibody (1:5000 diluted in PBST containing 1% skimmed milk) was added to each well and incubated for 1 h. After three washes with PBST, 100 mL of 1X TMB/H 2 O 2 solution was added and kept in dark for 3-5 min. Thereafter, the 96-well plate was read at 450 nm.

Western blotting
Cells lysates were prepared in ice-cold RIPA buffer with protease inhibitors. Lysates were collected in pre-chilled 1.5 mL tubes and incubated on ice for 15 min. Cell debris was removed by centrifugation at 15,000 Â g for 15 min at 4°C. Supernatants were stored as aliquots of 100 mL in 0.5 mL tubes at À20°C until use. Total protein was estimated by Lowry's method [4]. 30 mg of total protein was resolved by 10% SDS-PAGE and transferred to 0.45 m nitrocellulose membrane using semi-dry transfer method. Blotting was done either at constant current (140 mA) or voltage (16 V) for 80 min. After the transfer, blots were stained with Ponceau S and scanned. Ponceau S stain was removed by washing in PBS for 5 min followed by a 5 min wash with Tris buffered saline containing 0.05% Tween 20 (TBST). The blots were then blocked in 1% gelatin in TBST for 2 h at room temperature followed by overnight incubation at 4°C in 0.1% gelatin in TBST. Next day, the blots were brought to room temperature and rinsed with TBST. Blots were then probed with primary antibody diluted in 0.1% gelatin in TBST for 2 h at room temperature. Blots were washed for 30 min with TBST (3 Â 10 min) to remove unbound antibody. Blots were then incubated with HRP-tagged secondary antibody (1:5000 dilution in 0.1% gelatin in TBST) for 1 h followed by three TBST washes of 10 min each. Bands were visualized with enhanced chemiluminescence reagent (Santa Cruz Biotechnology, CA, USA). Chemidoc XRS þ system (BioRad) was used to capture the images.