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LearnPA1-518
antibody from Invitrogen Antibodies
Targeting: MTOR
FLJ44809, FRAP, FRAP1, FRAP2, RAFT1, RAPT1
Western blot
ImmunoprecipitationAntibody data
- Antibody Data
- Antigen structure
- References [2]
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- Validations
- Western blot [4]
- Immunocytochemistry [1]
- Other assay [2]
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- Product number
- PA1-518 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- mTOR Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Other
- Description
- 3-8% Tris-Acetate Gel is recommended for Western blot. PA1-518 detects a predominant band at ~290kD corresponding to mTOR. Other lower MW nonspecific bands of unknown identity were also detected at ~65kD and ~35kD in some cell lysates.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µg
- Concentration
- 1 mg/mL
- Storage
- -20°C
Submitted references Effect and molecular mechanism of mTOR inhibitor rapamycin on temozolomide-induced autophagic death of U251 glioma cells.
Bilirubin activates transcription of HIF-1α in human proximal tubular cells cultured in the physiologic oxygen content.
Li B, Zhou C, Yi L, Xu L, Xu M
Oncology letters 2018 Feb;15(2):2477-2484
Oncology letters 2018 Feb;15(2):2477-2484
Bilirubin activates transcription of HIF-1α in human proximal tubular cells cultured in the physiologic oxygen content.
Kim SG, Ahn SY, Lee ES, Kim S, Na KY, Chae DW, Chin HJ
Journal of Korean medical science 2014 Sep;29 Suppl 2(Suppl 2):S146-54
Journal of Korean medical science 2014 Sep;29 Suppl 2(Suppl 2):S146-54
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of mTOR was performed by loading 10 µg of the indicated whole cell lysates per well, and 5 µL of Spectra Multicolor High Range Protein Ladder (Product # 26626) onto a NuPAGE® 3-8% Tris-Acetate Gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 1 hour at room temperature. mTOR was detected at ~290 kDa after probing with an mTOR polyclonal antibody (Product # PA1-518) at a concentration of 6.5 µg/mL in StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) overnight at 4C on a rocking platform, washing in TBST, and probing with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of mTOR was performed by loading 10 µg of the indicated whole cell lysates per well, and 5 µL of Spectra Multicolor High Range Protein Ladder (Product # 26626) onto a NuPAGE® 3-8% Tris-Acetate Gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 1 hour at room temperature. mTOR was detected at ~290 kDa after probing with an mTOR polyclonal antibody (Product # PA1-518) at a concentration of 6.5 µg/mL in StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) overnight at 4C on a rocking platform, washing in TBST, and probing with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- CRISPR-Cas9 mediated genome editing ofmTOR (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayIDCRISPR814789_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of mTOR was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with mTOR Lentiviral sgRNA (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ 3 to 8%, Tris-Acetate, 1.0 mm, Mini Protein Gel (Product # EC66952BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-mTOR Polyclonal Antibody (Product # PA1-518) using 1:1000 dilution and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177 1:10000 dilution).Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076) . A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific tomTOR (Fig (b)).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-mTOR Polyclonal Antibody (Product # PA1-518) and a ~280kDa band corresponding to Serine/threonine-protein kinase mTOR was observed across cell lines and tissues tested . Whole cell extracts (30 µg lysate) of SiHa (Lane 1), K-562 (Lane 2), HEK-293 (Lane 3), MCF7 (Lane 4), MDA-MB-231 (Lane 5), HEL 92.1.7 (Lane 6), Hep G2 (Lane 7), Mouse Stomach (Lane 8), Rat Cerebellum (Lane 9), Mouse Heart (Lane 10) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2002) 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, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Reagent Kit (Product # WP20005).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescent analysis of mTOR (green) in HeLa cells. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 1% Blocker BSA (Product # 37525), each for 15 minutes at room temperature. Cells were stained with an mTOR polyclonal antibody (Product # PA1-518) at a concentration of 10 µg/mL in 1% Blocker BSA in PBS (right panel), or incubated in blocking buffer alone as a negative control (left panel) overnight at 4C, and then incubated with a Dylight 488-conjugated goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:1000 for 1 hour at room temperature. F-Actin (red) was stained with a DyLight 554-conjugated phalloidin control (Product # 21834) and nuclei (blue) were stained with DAPI (Product # 46190).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunoprecipitation of mTOR was performed on HeLa cells. Antigen-antibody complexes were formed by incubating 500 µg of HeLa whole cell lysate with 2 µg of mTOR polyclonal antibody (Product # PA1-518) overnight on a rocking platform at 4C. The immune complexes were captured on 50 µL of Protein A/G Agarose (Product # 20421), washed extensively, and eluted with Lane Marker Reducing Sample Buffer (Product # 39000). HeLa cell lysate (10 µg) was loaded as a positive control (right lane). Samples were resolved on a NuPAGE? 3-8% Tris-Acetate Gel, transferred to a PVDF membrane, and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 1 hour at room temperature. The membrane was probed with an mTOR polyclonal antibody (Product # PA1-518) at a concentration of 6.5 µg/mL in StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) overnight at 4C on a rocking platform, washed in TBST, and probed with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Supplement Fig. 1 The effect of bilirubin on the expression of mTOR and RAPTOR. Western blotting of cells cultured with (10 uM) or without H 2 O 2 or with (0.1 mg/dL) or without bilirubin. Human HK2 cells cultured under 5% oxygen condition for 1 hr. The vertical bar indicates 95% CI of the mean value.
