- PA5-77985 - Invitrogen Antibodies GFI1 antibody

Viryasova GM, Golenkina EA, Tatarskii VV Jr, Galkin II, Sud'ina GF, Soshnikova NV
MethodsX 2019;6:360-367

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of GFI-1 was achieved by transfecting Jurkat with GFI-1 specific siRNAs (Silencer® select Product # S5705, S5707). Western blot analysis (Fig. a) was performed using whole cell extracts of Jurkat prepared using RIPA lysis buffer supplemented with Universal Nuclease for Cell Lysis from the GFI-1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with GFI1 Polyclonal Antibody (Product # PA5-77985, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution). 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 GFI-1. Uncharacterized bands (*) at ~70 and ~140 kDa were observed in untransfected lysate and only ~140 kDa band was observed in scramble and siRNA lysates.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using GFI1 Polyclonal Antibody (Product # PA5-77985) and a 55 kDa band corresponding to GFI-1 was observed in Jurkat, HCT 116 and SW480 and not in T-47D and A549. Whole cell extracts (30 µg lysate) of Jurkat (Lane 1), HCT 116 (Lane 2), SW480 (Lane 3), T-47D (Lane 4) and A549 (Lane 5) prepared using RIPA lysis buffer supplemented with Universal Nuclease for Cell Lysis were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX), 10 well. Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Uncharacterized bands (*) were observed at ~70 and ~140 kDa in Jurkat cell lysate and only ~140 kDa uncharacterized band was observed in HCT 116, SW480 and T-47D cell lysates.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of GFI1 was performed in THP-1 cells fixed in 4% paraformaldehyde at RT for 15 min. Green: GFI1 Polyclonal Antibody (Product # PA5-77985) diluted at 1:500. Red: phalloidin, a cytoskeleton marker. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of GFI-1 was performed in Jurkat and T-47D 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 GFI1 Polyclonal Antibody (Product # PA5-77985) 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 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nuclear localization. Panel e represents no signal in the negative model (T-47D). Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Flow cytometry overlay diagrams demonstrate AlexaFluor488-labelled antibodies distribution in neutrophils after permeabilization with 70% EtOH, 90% MeOH or 0.1% saponin. (A) The efficiency of block of secondary Ab non-specific binding (fluorescence level of isotype control) depending on the permeabilization agents in human leukocytes, primary cells. (B) Isotype controls in case of different permeabilization agents in HL-60 cells. (C) Isotype controls in PMNLs after possible non-specific binding sites were blocked with different reagents: 1% BSA, 10% goat serum or both. (D) The alterations of GFI1 protein content during myeloid differentiation of cell line HL-60 with trans-retinoic acid (ATRA) and in human neutrophils, samples were prepared according to the Protocol. (E) The median fluorescence intensity data for GFI1 protein. Values indicate the mean +- SD from three independent experiments. ** p < 0.01, *** p < 0.005 compared to the GFI1 content in HL-60. For all pairs Isotype-GFI1b p-value is less than 0.005 (not mentioned on the diagram. Statistical analysis was done with 2-way ANOVA, Holm-Sidak's multiple comparison test. Fig. 2

PA5-77985