MA5-12858

antibody from Invitrogen Antibodies

Targeting: PHB PHB1

Provider product page for MA5-12858

Western blot

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [68]
Comments [0]
Validations
Western blot [3]
Immunohistochemistry [2]
Other assay [7]

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Product number: MA5-12858 - Provider product page
Provider: Invitrogen Antibodies
Product name: Prohibitin Monoclonal Antibody (II-14-10)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA5-12858 targets Prohibitin in immunofluorescence, immunohistochemistry (paraffin), and Western blot applications and shows reactivity with Chicken, Human, mouse, Porcine, and Rat samples.
Antibody clone number: II-14-10
Concentration: 0.2 mg/mL

PHB protein structure - MA5-12858 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of Prohibitin using Prohibitin Monoclonal Antibody (Product # MA5-12858) on PC12 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Prohibitin was achieved by transfecting HEPG2 cells with Prohibitin specific siRNAs (Silencer® select Product # s10424 and s10425). Western blot analysis (Fig a) was performed using Membrane extract from the Prohibitin knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-Prohibitin Mouse Monoclonal Antibody (Product # MA5-12858, 0.2 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to Prohibitin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg of lysate) of Caco-2 (Lane 1), Caco-2 treated with H2O2 (100uM for 1 hr) (Lane2), Neuro-2a (Lane 3), Neuro-2a treated with H2O2 (100uM for 1 hr) (Lane 4), COS-7 (Lane 5), COS-7 treated with H2O2 (100uM for 1 hr) (Lane 6) and HepG2 (Lane 7). The blots were probed with anti-Prohibitin Mouse Monoclonal Antibody (Product # MA5-12858, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 30 kDa band corresponding to Prohibitin was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Prohibitin using Prohibitin Monoclonal Antibody (Product # MA5-12858) on denatured Mouse MAD109 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Prohibitin using Prohibitin Monoclonal Antibody (Product # MA5-12858) on Native Mouse MAD109 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIGURE 2. Submitochondrial localization of the mitochondrial pseudouridine synthases. A , confocal analysis of each of the five mitochondrial pseudouridine synthases. 143B cells were transfected with expression plasmids encoding the FLAG-tagged proteins indicated. Mitochondria were stained using MitoTracker Deep Red FM (in blue ). The cells were immunolabeled with anti-FLAG and anti-FASTKD2 as an MRG-specific marker. White boxes indicate the regions shown at higher magnification. Scale bars are 10 mum. B , subcellular fractionation of 143B cells and analysis by Western blotting show enrichment for the endogenous RPUSD4 in the mitochondrial fraction. Total cell lysate ( T ), mitochondrial ( M ), and cytosolic ( C ) fractions were assessed for purity using beta-tubulin and VDAC1 as cytosolic and mitochondrial markers, respectively. C , immunoblotting analysis of total isolated 143B mitochondria ( T ) or the supernatant ( S ) and pellet ( P ) fractions following alkaline sodium carbonate ( Na 2 CO 3 ) extraction. mtHSP70 and prohibitin ( PHB ) are markers for the mitochondrial matrix and mitochondrial membranes, respectively. D , immunoblot analysis of isolated 143B mitochondria after proteinase K ( PK ) accessibility test. Mitochondria were left untreated, swollen to rupture of the outer membrane, or lysed in Triton X-100 prior to treatment with proteinase K. mtHSP70 is a mitochondrial matrix protein, Smac/DIABLO is an intermembrane space protein, and TOM20 is a mitochondrial o

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7. Differential localization of MTCH2 isoforms. A, plasmids expressing FLAG-HA-tagged isoforms of MTCH2 were transfected in HEK293 cells. Mitochondrial and cytoplasmic fractions from these cells were used for Western blotting. VDAC and alpha-tubulin were used as markers for mitochondria and cytoplasm, respectively. B, mitochondrial localization of endogenous MTCH2 and MTCH2x in HEK293 cells. Prohibitin was used as a marker for mitochondria. C, confocal fluorescence microscopy images showing cellular localization of GFP-tagged MTCH2 isoforms in primary bovine aortic endothelial cells. MitoTracker Red CMXRos was used to stain mitochondria. Percentage of GFP-positive cells in case of GFP-MTCH2xx was less compared with GFP-MTCH2 and GFP-MTCH2x. One such cell is shown. Scale bar , 10 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 PHB is bound directly to Mlph in Melan-a cells. A IB analysis for Rab27a, PHB, Mlph, and myosin-Va of Ashen Melan-a cells. B COS7 monkey kidney cells were transfected with PHB and Mlph plasmids. Anti-histidine immunoprecipitated (IP) were performed for 3 h at 4 degC followed by western blot analysis to check co-IP (WB). Whole cell lysates were analyzed for total protein levels.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5. Up-scaling of PEG-precipitation. EVs from 360 mL HEK293T-CD63-eGFP cell conditioned media were prepared by PEG precipitation. The particle concentration per fraction ( n = 3; SD, (a) and their average size distribution ( n = 3; SD, (b) were assessed by NTA (ZetaView). The purity of the obtained samples was determined as particle numbers - measured by NTA - per mg protein content - measured by the BCA assay (c). The presence of the EV marker proteins HSP70, Tsg101, CD63, CD81 and CD9 within the individual PEG samples was analysed by western blot. Additionally, the content of BSA, Syntenin and Prohibitin within the final samples was compared; 5 ug proteins were loaded per lane (d). To test for potential impacts on the physiology of the obtained EVs, uptake experiments were performed: 30-50 ug of the obtained samples were supplemented to the cell culture media of N-KM cells. After 14-16 h pictures were taken, then, the amount of the eGFP labelling was quantified as the mean fluorescence intensity (MFI) by flow cytometry (e). Scale bars 20 um.