All lanes : Anti-Transferrin antibody [HTF-14] (ab769)

Lane 1 : Human Transferrin – 8 µl (reducing conditions) at 5 µg
Lane 2 : HumanTransferrin – 5 µl (reducing conditions) at 3 µg
Lane 3 : Human Transferrin – 3 µl (reducing conditions) at 1 µg
Lane 4 : Human Transferrin – 3 µl (non reducing conditions) at 1 µg
Lane 5 : Human Transferrin – 5 µl (non reducing conditions) at 3 µg
Lane 6 : Human Transferrin - 8 µl (non reducing conditions) at 5 µg

The sample (commercial human transferrin) (1µg/ml in 1x PBS) was mixed with 4x loading buffer (non reducing) or with Laemmli buffer (reducing) and warmed for 3 minutes.

Volume of sample:5; 3; 1 µl of sample + 3; 2; 2 µl of buffer

SDS Page: 12% AA-gel, U=180V, 90 minutes, volume of loading sample:8; 5;3 µl

Marker – 3 µl

WB: Nitrocelulose membrane (8x5.5 cm), I=35mA, 90minutes

Detection:

Blocking solution: 1xPBS/5% BSA, 4°C, overnight

Primary Ab: membrane was incubated in 5 ml DMEM/10%FTS + 10 µl HTF-14 (primary Ab) (60 minutes, room temperature)

Washing solution: 1x PBS/0.1% Tween

Secondary Ab: IRDye^® 800CW Goat anti-mouse IgG1-specific, dilution 1/5000, diluting solution – 5% BSA/1x PBS, incubation for 60 minutes, low temperature.

Detection: Li-COR odyssey

No reducing agent should be added to the sample buffer. The sample can be denatured and processed by common Western blotting protocols, but the disulfide bonds must not be reduced.

Standard curve for Transferrin (Analyte: ab83560); dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal [HTF-14] to Transferrin (ab769) at 1µg/ml and Detector Antibody Rabbit polyclonal to Transferrin (ab9538) at 0.5µg/ml.