phb2 primary antibody  (Proteintech)

Journal: bioRxiv

Article Title: Characterization of prohibitins as novel target to block asexual and sexual stage growth of Plasmodium falciparum

doi: 10.1101/2022.06.02.494630

Figure Lengend Snippet: (A) Diagrammatic representation of Pf PHB1 and Pf PHB2, showing their SPFH (PHB/Band-7) domain (in green), expressed as recombinant proteins. (B, C) Coomassie-stained SDS-PAGE representing purified recombinant Pf PHB1 and Pf PHB2 tagged with GST (B) and MBP (C). Selected Pf PHB1 and Pf PHB2 coding regions cloned in pGEX-4T 1 and pMAL TM c5X vectors, after induction with appropriate concentration of Isopropyl β-d-1-thiogalactopyranoside (IPTG) were purified with affinity-binding methods. GST-tagged Pf PHB1 (∼56.5 KDa) and Pf PHB2 (∼60.7 KDa) recombinant proteins were purified with glutathione-sepharose beads. While, the MBP-tagged Pf PHB1 (∼68 KDa) and Pf PHB2 (∼74 KDa) recombinant proteins were purified from inclusion bodies using amylose-resins. (D, E) Western blot analysis of purified recombinant proteins to validate their purity and specificity. The proteins were probed with mice anti-GST (dilution 1:1000) (D) and anti-MBP (dilution 1:5000) (E) primary antibodies for respective recombinant proteins. After washing, the blots were probed with anti-mice secondary antibody (dilution 1:5000). The blots were developed to detect specific bands of the purified recombinant proteins. To raise specific antibodies, Balb/c mice were immunized with 100 μg of each recombinant protein.

Article Snippet: The binding of Pf PHB1 and Pf PHB2 primary antibodies was detected by incubating the samples with monoclonal Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (dilution 1:1000; Molecular Probes, USA) for 1 hour at room temperature.

Techniques: Recombinant, Staining, SDS Page, Purification, Clone Assay, Concentration Assay, Binding Assay, Western Blot

Journal: bioRxiv

Article Title: Characterization of prohibitins as novel target to block asexual and sexual stage growth of Plasmodium falciparum

doi: 10.1101/2022.06.02.494630

Figure Lengend Snippet: (A) Parasite lysates prepared from both asexual (rings, trophozoites and schizonts) and sexual (immature and mature gametocytes (Gc)) stages and gametocytes at 30 minutes p.a. (aGC) were subjected to Western blot analyses. Immunolabelling was performed with polyclonal mouse anti- Pf PHB1 (dilution 1:500; ∼30.5 KDa) and anti- Pf PHB-2 (dilution 1:250; ∼34.7 KDa) antisera to depict expression profile of the proteins. Equal loading was confirmed using a polyclonal mouse anti- Pf 39 antiserum (dilution 1:1000; ∼39 KDa). Lysate of uninfected erythrocytes was used as a negative control. (B) Immunofluorescence assay was performed as a negative control on asexual (schizonts) and sexual (Stage V) stages. Parasites were incubated with non-immunized mice sera (NMS; dilution 1:100; green) and counter labelled with rabbit antibodies directed against-merozoite surface protein-1 ( Pf MSP-1; dilution 1:200) and Pf s230 (dilution 1:200) as indicated (red). Nuclei were highlighted with Hoechst33342 nuclear stain (blue). (C, D) Immunofluoroscence assays to decipher expression and sub-cellular localization of Pf PHB1 (C) and Pf PHB2 (D) in asexual (trophozoites and schizonts) and sexual (stage III-V) stages of the parasite via immunolabelling with polyclonal mouse anti- Pf PHB1 (dilution 1:50) and anti- Pf PHB2 (dilution 1:50) antisera (green). Asexual stages were highlighted with polyclonal rabbit anti- Pf MSP-1 (dilution 1:200) antibody, whereas, gametocyte stages were marked with rabbit anti- Pf s230 (dilution 1:200) antibody (red). Nuclei were marked with Hoechst33342 nuclear stain (blue). Bar, 10 μm. Results are representative of three independent experiments.

Article Snippet: The binding of Pf PHB1 and Pf PHB2 primary antibodies was detected by incubating the samples with monoclonal Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (dilution 1:1000; Molecular Probes, USA) for 1 hour at room temperature.

Techniques: Western Blot, Expressing, Negative Control, Immunofluorescence, Incubation, Staining

Journal: bioRxiv

Article Title: Characterization of prohibitins as novel target to block asexual and sexual stage growth of Plasmodium falciparum

doi: 10.1101/2022.06.02.494630

Figure Lengend Snippet: (A) Sub-cellular localization of Pf PHB1 and Pf PHB2 were detected in cytosolic and organellar fractions of enriched asexual parasites (trophozoites) which were subjected to Western blot analysis probed with their respective mice antisera. Bands were detected at ∼30.5 KDa (A) and ∼34.7 KDa (B) for Pf PHB1 and Pf PHB2, respectively, in organellar fractions. Rabbit antibody against H4Kac4 (dilution 1:1000) detecting acetylated histone H4 (∼11 and ∼13 KDa) was used as organellar fraction control while rabbit anti-NapL antisera (1:500) with size at ∼40.3 KDa. (C) Asexual stage (trophozoite) lysate of the parasite was used to extract soluble, peripheral, integral membrane and insoluble protein fractions. The samples were subjected to Western blot and immunolabelled with mouse anti- Pf PHB1 antisera to detect Pf PHB-1 (∼30.5 KDa). Rabbit anti-H4Kac4 (dilution 1:1000) antisera (∼11 and ∼13 KDa), mouse anti- Pf 39 (dilution 1:1000) antisera (∼39 KDa) and rabbit anti- Pf GAP45 (dilution 1:100) antisera (∼45 KDa).

Article Snippet: The binding of Pf PHB1 and Pf PHB2 primary antibodies was detected by incubating the samples with monoclonal Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (dilution 1:1000; Molecular Probes, USA) for 1 hour at room temperature.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Characterization of prohibitins as novel target to block asexual and sexual stage growth of Plasmodium falciparum

doi: 10.1101/2022.06.02.494630

Figure Lengend Snippet: (A, B) Western blot analysis representing pulling down of Pf PHB1 (A) and Pf PHB2 (B) from parasite lysate using recombinant Pf PHB2 and Pf PHB1 proteins, respectively. Briefly, the recombinant proteins were incubated with GST-beads in binding buffer followed by incubation with the parasite lysate (pRBCs). Incubation with uninfected RBCs (RBCs) was used as a control. The unbound proteins were removed through centrifugation and the proteins bound to the GST-beads were eluted using 10 mM Glutathione. The blots were probed with mice antisera against Pf PHB1 (dilution 1:500; ∼30.5 KDa) and Pf PHB2 (dilution 1:250; ∼34.7 KDa) followed by rabbit anti-mice HRP-conjugated secondary antibody (dilution 1:2000). (C, D) Dissociation (C) and dose-response (D) curves representing interaction analysis of Pf PHB1 with Pf PHB2 using MST. 10 µM Pf PHB2 recombinant protein, diluted in 1X PBS/0.01% Tween20, with decreasing concentrations were titrated against the constant concentration of the labelled Pf PHB1 recombinant protein. K d value was determined to be 135.5 ± 10.5 nM for interaction of Pf PHB1 with Pf PHB2.

Article Snippet: The binding of Pf PHB1 and Pf PHB2 primary antibodies was detected by incubating the samples with monoclonal Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (dilution 1:1000; Molecular Probes, USA) for 1 hour at room temperature.

Techniques: Western Blot, Recombinant, Incubation, Binding Assay, Centrifugation, Concentration Assay

Journal: bioRxiv

Article Title: Characterization of prohibitins as novel target to block asexual and sexual stage growth of Plasmodium falciparum

doi: 10.1101/2022.06.02.494630

Figure Lengend Snippet: (A-D) Dissociation (A,C) and dose-response (B, D) curves representing interaction analysis of recombinant proteins Pf PHB1 (A,B) and Pf PHB2 (C,D) with Roc-A through MST. Labelled Pf PHB1 and Pf PHB2 recombinant proteins, diluted in 1X PBS/0.01% Tween20, were titrated against decreasing concentrations 100 µM Roc-A. K d values were determined to be 1.37 ± 0.6 µM and 0.683 ± 0.021 µM for interaction of Pf PHB1 and Pf PHB2, respectively. (E,G) Western blots representing thermo stability analysis to validate interaction of Roc-A with native Pf PHB1 and Pf PHB2. To serve this purpose, trophozoite stage parasites were treated with 50 µM Roc-A followed by incubation for 4 hours along with the untreated control. After harvesting the parasites, cells were lysed and the pellets were resuspended in RIPA lysis buffer followed by heating the samples at different temperatures (40, 60 and 80 °C) for 6 minutes. Following centrifugation at 10,000 rpm for 40 minutes at 4°C, supernatant was transferred to new tubes and were subjected to Western blot to evaluate the change in protein stability in the presence and absence of Roc-A. (F,H) Histogram plot representing densitometry analysis of the bands performed with ImageJ software for the TSA of Pf PHB1 and Pf PHB2 with Roc-A.

Article Snippet: The binding of Pf PHB1 and Pf PHB2 primary antibodies was detected by incubating the samples with monoclonal Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (dilution 1:1000; Molecular Probes, USA) for 1 hour at room temperature.

Techniques: Recombinant, Western Blot, Incubation, Lysis, Centrifugation, Software

Journal: bioRxiv

Article Title: Characterization of prohibitins as novel target to block asexual and sexual stage growth of Plasmodium falciparum

doi: 10.1101/2022.06.02.494630

Figure Lengend Snippet: (A, B) Line graph reflecting growth curve of S. cerevisiae BY4742 wild type ( Sc WT), phb1Δ and phb2Δ yeast mutants, and yeast complemented phb1Δ- Pf phb1-p416 GPD, phb2Δ- Pf phb2-p416 GPD strains in the absence (A) and presence (B) of EtBr. The yeast cells were pre-cultured with/without EtBr and later diluted to initial A 600 =0.05 with YPD medium to be used further as the main culture. After every 20 hours, the A 600 was measured spectrophotometrically to quantitate the growth of EtBr treated and untreated yeast cells. (C, D) Spot assay showing growth of yeast cells spotted following 3 days of incubation. Each culture was harvested and adjusted to A 600 = 0.5 using sterile YPD medium. The particular solution was then serially diluted 10-times and each dilution suspension was then spot on solid YPD medium and incubated for two days at 30 °C.

Article Snippet: The binding of Pf PHB1 and Pf PHB2 primary antibodies was detected by incubating the samples with monoclonal Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (dilution 1:1000; Molecular Probes, USA) for 1 hour at room temperature.

Techniques: Cell Culture, Spot Test, Incubation

Journal: bioRxiv

Article Title: Characterization of prohibitins as novel target to block asexual and sexual stage growth of Plasmodium falciparum

doi: 10.1101/2022.06.02.494630

Figure Lengend Snippet: (A, B) Line graph showcasing growth of yeast cells of S. cerevisiae BY4742 wild type ( Sc WT), phb1Δ and phb2Δ yeast mutants, and yeast complemented phb1Δ- Pf phb1-p416 GPD (A), phb2Δ- Pf phb2-p416 GPD (B) strains in the absence and/or presence of EtBr and/or Roc-A. Roc-A and EtBr were used at a concentration of 6 µM and 25 µg/ml, respectively. The yeast cells were pre-cultured with/without EtBr and later diluted to initial A 600 =0.05 with YPD medium to be used further as the main culture in the presence and absence of Roc-A. After every 20 hours, the A 600 was measured spectrophotometrically to quantitate the growth of yeast cells. (C, D) Spot assay showing growth of yeast cells spotted following 2 days of incubation. Each culture was harvested and adjusted to A 600 = 0.5 using sterile YPD medium. The particular solution was then serially diluted 10-times and each dilution suspension was then spot on solid YPD medium and incubated for two days at 30 °C.

Article Snippet: The binding of Pf PHB1 and Pf PHB2 primary antibodies was detected by incubating the samples with monoclonal Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (dilution 1:1000; Molecular Probes, USA) for 1 hour at room temperature.

Techniques: Concentration Assay, Cell Culture, Spot Test, Incubation