Cell lysates were separated on NuPAGE® 4–12% Bis-Tris gel at 200 V for 45 min in 1X NuPAGE® MES SDS running buffer (Life Technologies) and electrophoretically transferred to Amersham Hybond-P PVDF membrane (GE Healthcare) at 30 V for 60 min in 1X NuPAGE® transfer buffer (Life Technologies). PVDF membranes were blocked with 5%-milk-TBST at RT. The membranes were incubated with rabbit polyclonal anti-AKR1C3 antibody (1:2’000, Thermo Scientific), followed by incubation with anti-rabbit IgG HRP antibody (1:5’000, GE Healthcare). The protein was detected using a Pierce® ECL Western Blotting Substrate (Thermo Scientific). Treated membranes were exposed to X-ray film. The membrane was stripped, incubated with anti-actin antibody (1:1'000, Sigma Aldrich) and analyzed again. Protein bands were quantified with ImageJ software [39 ], and were adjusted for corresponding actin loading control.
Anti rabbit igg hrp antibody
Manufactured by GE Healthcare
The Anti-rabbit IgG HRP antibody is a laboratory reagent used to detect and quantify rabbit immunoglobulin G (IgG) in various experimental applications. It is conjugated with horseradish peroxidase (HRP), an enzyme that catalyzes a color-producing reaction, allowing for the visualization and measurement of target proteins in samples.
Automatically generated - may contain errors
Lab products found in correlation
Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Amersham hybond p pvdf membrane, by GE Healthcare (1 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Novex 4 12 tris glycine gel, by Thermo Fisher Scientific (1 mentions)
P akt s473 d9e, by Cell Signaling Technology (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ethidium bromide, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v1, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 2, by Roche (1 mentions)
Bkm120, by Selleck Chemicals (1 mentions)
Pakt ser473 d9e, by Cell Signaling Technology (1 mentions)
P akt s473 alexa fluor 647 d9e, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Human gamma globulin fraction 2, by ICN Biomedicals (1 mentions)
3 protocols using anti rabbit igg hrp antibody
1
Western Blot Analysis of AKR1C3 Protein
2
Western Blot Analysis of p-AKT and AKT
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Cells were washed and resuspended in 2× SDS sample buffer at 106 cells/100 µl and boiled for 5 min. Samples were separated on Novex 4–12% Tris-Glycine gel (Invitrogen) and transferred to a PVDF membrane (Immobilon-P; EMD Millipore). Proteins were detected with the following primary antibodies: p-AKT(S473) (D9E) and AKT (5G3; Cell Signaling Technology). An anti–rabbit IgG-HRP antibody (GE Healthcare) was used as the secondary antibody.
3
Immune Cell Signaling and Activation
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Doxycycline, puromycin, ethidium bromide, and Lipofectamine 2000 were purchased from Invitrogen. Recombinant human CCL17/TARC and recombinant human CCL22/MDC were purchased from R&D Systems. Human recombinant IL2 was obtained from Roche. Anti–human CCR4 antibodies (clone 1G1) conjugated to PE and Alexa Fluor 647 fluors were purchased from BD. The following antibodies were purchased from Cell Signaling Technology: Akt, P-Akt (Ser473; D9E), P-Akt (S473)–Alexa Fluor 647 (D9E), and Alexa Fluor 647–conjugated isotype control antibody (DA1E). Anti–rabbit IgG-HRP antibody was purchased from GE Healthcare. CD8a(Lyt 2) microbeads were purchased from Miltenyi Biotec. BKM120 was purchased from Selleck Chemicals. PTX was purchased from Sigma-Aldrich. Big Dye Terminator v1.1 was purchased from Applied Biosystems. Human gamma globulin fraction II was purchased from ICN Biomedicals Inc. Paraformaldehyde was purchased from Electron Microscopy Sciences.
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