Figure 1.
Protein profile of eluted fractions containing 31 kDa as a major eluted kidney bean protein.
Kidney bean extract was subjected to anion exchange chromatography (A). Lane 1−5: fractions (10 to 14). Protein profile of gel filtration eluted fractions containing 31 kDa as a major eluted protein (B). Lane 1−3: fractions (17 to 19). Elution profile of purified 31 kDa protein after HPLC (C). Lane 1: purified protein after silver staining on SDS-PAGE, lane 2: immunoblot of purified protein using kidney bean hypersensitive pooled patients’ sera.
Table 1.
SPT and specific IgE among the patients (1−25) and controls (C1−C5) against raw kidney bean extract and 31 kDa protein.
Figure 2.
IgE binding of purified 31 kDa protein by immunoblotting.
Lane 1−25: with individual patients’ sera and lane C: purified protein probed with normal human sera (control).
Figure 3.
Histamine released from stripped basophils re-sensitized with individual patients’ sera (n = 15) on challenge with kidney bean extract and 31 kDa purified protein, separately.
Lane C1−C5: controls (A). Scatter plot of the correlation analysis between % histamine released by patients (n = 15) and controls on challenge with crude kidney bean extract and purified protein (B). 2-DE of purified 31 kDa protein showing two close spots (1 & 2) at the same molecular weight. Both the spots were identified as PHA-E after mass spectrometric analysis (C).
Table 2.
Peptide mass fingerprint database search of selected spots from 2-DE of purified 31 kDa kidney bean proteins.
Figure 4.
Glycoprotein detection by Periodic Acid Schiff’s (PAS) staining.
Lane 1: kidney bean extract, lane 2: purified 31 kDa protein (A). Immunoblot of purified protein after periodate treatment. The 31 kDa protein was electro-transfered onto nitrocellulose membrane and then periodate oxidation was done. The strip was washed, blocked with 3% defatted milk and immunoblotted with pooled patients’ sera. Lane 1: 31 kDa protein (untreated), lane 2: 31 kDa protein after periodate treatment (B). Hemagglutination assay of 31 kDa protein, PHA (positive control) and PBS (negative control). Both 31 kDa and PHA formed uniform reddish color across the well with a minimum concentration of 15.62 µg/ml (C). Agglutination of human erythrocytes using purified protein and PHA (Sigma) (D).
Figure 5.
SDS-PAGE profile and immunoblot of 31 kDa kidney bean protein.
Lane 1 : untreated, Lane 2−5 : boiled at 100°C for 15, 30, 45 and 60 min, Lane M: molecular weight markers. Protein fractions were stained with Coomassie brilliant blue (A) and immunoblotted (B). SGF digestion of 31 kDa protein Lane M:molecular weight markers, Lane 1: 31 kDa protein (undigested), lanes 2−8: 31 kDa protein incubated in SGF for 0.5, 1, 5, 10, 15, 30 and 60 min, lane 9: pepsin. The digested protein was electrophoresed on SDS-PAGE and visualized by CBB staining (C).
Figure 6.
IgE ELISA inhibition of kidney bean extract and 31 kDa protein.
Kidney bean protein and extracts of different legumes were used as inhibitor (A). Kidney bean positive patients’ pooled sera (1∶10 v/v) was preincubated with 5, 10, 50, 100, 1000, 10000 ng of inhibitor. ELISA was carried out on solid phase coated kidney bean extract (2 µg/100 µl/well) or 31 kDa protein (500 ng/100 µl/well) and preincubated sera. Immunoblot inhibition of kidney bean extract with 31 kDa protein (B). Kidney bean positive pooled patients’ sera was preincubated with 100 µg of 31 kDa protein (lane 2). Immunoblot using kidney bean positive pooled patients’ sera without inhibitor was used as a control (lane 1).