- MA5-14733 - Invitrogen Antibodies FTL antibody

Ohshima T, Yamamoto H, Sakamaki Y, Saito C, Mizushima N
The Journal of cell biology 2022 Oct 3;221(10)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Sandwich ELISA of Ferritin was performed by coating wells of a 96-well plate with 100 µL of an Ferritin duck polyclonal antibody (Product # D0107-2D) diluted to a concentration of 1 µg/mL in carbonate/bicarbonate buffer (Product # 28382) overnight at 4C. Wells were blocked with 100 µL of StartingBlock T20 (TBS) Blocking Buffer (Product # 28382) for 2 hours, and 100 µL of recombinant human ferritin light polypeptide (FTL) standards ranging from 1.6-1000 ng/mL were incubated for 1 hour at room temperature. The plate was washed with 1X TBST (Product # 28360), and 100 µL per well of an ferritin mouse monoclonal antibody (Product # MA5-14733) diluted to a concentration of 1 µg/mL was added to each well for 1 hour at room temperature. The plate was washed, and 100 µL per well of a Streptavidin-HRP (Product # 21126) was incubated for 30 minutes. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028), followed by Stop Solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Ferritin (FTL, green) in HepG2 cells. The cells were fixed with formalin for 15 minutes, permeabilized with 0.1% Triton X-100 in TBS for 10 minutes, and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were stained with or without Ferritin monoclonal antibody (Product # MA5-14733), at a dilution of 1:200 for at least 1 hour at room temperature, and then incubated with a DyLight 488 goat anti-mouse secondary antibody (Product # 35502) at a dilution of 1:500 for 30 minutes at room temperature (both panels, green). F-Actin (both panels, red) was stained with DyLight 554 Phalloidin (Product # 21834) and nuclei (both panels, blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Sandwich ELISA of Ferritin was performed by coating wells of a 96-well plate with 100 µL of an Ferritin duck polyclonal antibody (Product # D0107-2D) diluted to a concentration of 1 µg/mL in carbonate/bicarbonate buffer (Product # 28382) overnight at 4C. Wells were blocked with 100 µL of StartingBlock T20 (TBS) Blocking Buffer (Product # 28382) for 2 hours, and 100 µL of recombinant human ferritin light polypeptide (FTL) standards ranging from 1.6-1000 ng/mL were incubated for 1 hour at room temperature. The plate was washed with 1X TBST (Product # 28360), and 100 µL per well of an ferritin mouse monoclonal antibody (Product # MA5-14733) diluted to a concentration of 1 µg/mL was added to each well for 1 hour at room temperature. The plate was washed, and 100 µL per well of a Streptavidin-HRP (Product # 21126) was incubated for 30 minutes. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028), followed by Stop Solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Direct ELISA analysis of Ferritin was performed by coating wells of a 96-well plate with 100 µL per well of Human Ferritin or BSA diluted in carbonate/bicarbonate buffer (Product # 28382), starting at a concentration of 1 µg/mL and serially diluting 2-fold to a concentration of 2 ng/mL, overnight at 4C. Wells of the plate were washed, blocked with StartingBlock blocking buffer (Product # 37538), and incubated with 100 µL per well of a mouse anti-ferritin monoclonal antibody (Product # MA5-14733) at a concentration of 1 µg/mL for 1 hour at room temperature. The plate was washed, then incubated with 100 µL per well of an HRP-conjugated goat anti-mouse IgG secondary antibody (Product # 31430) at a dilution of 1:25000 for 30 minutes at room temperature. Detection was performed using 1-Step Ultra TMB substrate (Product # 34028) for 10 minutes at room temperature in the dark. The reaction was stopped with 0.16M sulfuric acid, and absorbances were read on a spectrophotometer at 450-550 nm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Sandwich ELISA of Ferritin was performed by coating wells of a 96-well plate with 100 µL of an Ferritin mouse monoclonal antibody (Product # MA5-14733) diluted to a concentration of 1 µg/mL in carbonate/bicarbonate buffer (Product # 28382) overnight at 4C. Wells were blocked with 100 µL of StartingBlock T20 (TBS) Blocking Buffer (Product # 28382) for 2 hours, and 100 µL of recombinant human ferritin light polypeptide (FTL) standards ranging from 1.6-1000 ng/mL (A) or 100 µL of diluted normal human serum or diluted normal human plasma (B) were incubated for 1 hour at room temperature. The plate was washed with 1X TBST (Product # 28360), and 100 µL per well of an biotin-conjugated ferritin rabbit polyclonal antibody (Product # PA1-26882) diluted to a concentration of 1 µg/mL was added to each well for 1 hour at room temperature. The plate was washed, and 100 µL per well of a Streptavidin-HRP (Product # 21126) was incubated for 30 minutes. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028), followed by Stop Solution (Product # N600). Absorbances were read on a spectrophotometer at 450-550 nm.

MA5-14733