PD048
Lot 006~
Page 1For Research Use Only.
Not for use in diagnostic procedures.

Anti-OsTIR1 pAb
CODE No.

PD048

CLONALITY
ISOTYPEQUANTITY

PolyclonalRabbit IgG
100 µL

SOURCEIMMUNOGEN
REACTIVITY
FORMULATIONSTORAGE

Purified IgG from rabbit serum
Recombinant full-length Oryza sativa Transport inhibitor response 1 (OsTIR1) protein
This antibody reacts with Oryza sativa Transport inhibitor response 1 (OsTIR1).
PBS containing 50% Glycerol (pH 7.2). No preservative is contained.
This antibody solution is stable for one year from the date of purchase when stored at -20°C.

APPLICATION-CONFIRMED
Western blotting

1:1,000

APPLICATION-REPORTED
Immunohistochemistry

REFERENCE

Reference 2)

1) Natsume, T., et al., Cell Rep. 15, 210-218 (2016) [WB]
2) Yesbolatova A et al., Nat Commun. 11, 5701 (2020) [IC]For more information, please visit our web site at https://ruo.mbl.co.jp/.

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.

URL https://ruo.mbl.co.jp e-mail support@mbl.co.jp

PD048
Lot 006~
Page 2The descriptions of the following protocols are examples. Each user should determine the appropriate condition.

SDS-PAGE & Western blotting1) Culture the cells in the appropriate condition on a 6-well plate until they reach about 90% confluence.
2) Wash the cells with PBS and resuspend them with 50 µL of RIPA buffer [25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1%NP-40, 1% sodium deoxycholate, 0.1% SDS]. Transfer the lysate to a tube. Incubate on ice for 30 min.
3) Centrifuge the tube at 12,000 xg for 20 min. and transfer the supernatant to another tube.
4) Mix the supernatant with equal volume of Laemmli’s sample buffer.
5) Boil the samples for 5 min. and centrifuge. Load 5 µL of the sample per lane in a 1-mm-thick SDS-polyacrylamide gel (10%acrylamide) for electrophoresis.
6) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hr. in a semi-dry transfer system(Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% methanol). See the manufacturer's manual for precise transferprocedure.
7) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) overnight at 4°C.
8) Incubate the membrane with primary antibody diluted with 5% skimmed milk (in PBS, pH 7.2) as suggested in theAPPLICATIONS for 1 hr. at room temperature. (The concentration of antibody will depend on the conditions.)
9) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 min. x 3).
10) Incubate the membrane with the 1:10,000 Anti-IgG (H+L chain) (Rabbit) pAb-HRP (MBL; code no. 458) diluted with 5%
skimmed milk (in PBS, pH 7.2) for 1 hr. at room temperature.
11) Wash the membrane with PBS-T (5 min. x 3)
12) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 min. Remove extra
reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap.
13) Expose to an X-ray film in a dark room for 1 min. Develop the film as usual. The condition for exposure and development
may vary.

(kDa)

12

150
100
75
503725Western blotting analysis of OsTIR1

Sample: HCT116 Tet-OsTIR1
Lane 1: Untreated
Lane 2: Treated with 0.2 µg/µL of doxycycline for 24 hImmunoblotted with Anti-OsTIR1 pAb (PD048)
Sample was kindly provided by Dr. Masato Kanemaki. (National Institute of Genetics)
231226-4