Antibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [3]
- Immunocytochemistry [2]
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Validation data
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- Product number
- PA5-30419 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Mitofilin Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant protein fragment
- Description
- Recommended positive controls: U87-MG, MCF-7.
- Concentration
- 1 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot using Mitofilin Polyclonal Antibody (Product # PA5-30419). Sample (30 µg of whole cell lysate). Lane A: U87-MG . 7.5% SDS PAGE. Mitofilin Polyclonal Antibody (Product # PA5-30419) diluted at 1:1,000.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of Mitofilin was achieved by transfecting HeLa with Mitofilin specific siRNAs (Silencer® select Product # S21634, S21635). Western blot analysis (Fig. a) was performed using whole cell extracts from the Mitofilin knockdown cells (lane 2) and non-targeting scrambled siRNA transfected cells (lane 1). The blot was probed with Mitofilin Polyclonal Antibody (Product # PA5-30419, 1:1000) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10,000). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Mitofilin.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Mitofilin Polyclonal Antibody (Product # PA5-30419) and a 83 kDa band corresponding to Mitofilin was observed across all tested cell line lysates, along with an uncharacterized band at 60 kDa. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), A549 (Lane 2), HEK-293 (Lane 3), U-2 OS (Lane 4) and HeLa (Lane 5) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX), 12 well. Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10,000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Mitofilin Polyclonal Antibody detects Mitofilin protein at mitochondria by immunofluorescent analysis. Sample: MCF-7 cells were fixed in 2% paraformaldehyde/culture medium at 37ºC for 30 min. Green: Mitofilin protein stained by Mitofilin Polyclonal Antibody (Product # PA5-30419) diluted at 1:500. Blue: Hoechst 33342 staining.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of Mitofilin was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Mitofilin Polyclonal Antibody (Product # PA5-30419) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790, 1:2000) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: blue) were stained with Hoechst33342 (Product # H1399). F-actin (Panel c: Red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product # A30107, 1:2000). Panel d represents the merged image showing cytoplasmic (mitochondria-like) localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al. / Methods 115 (2017) 28–41).