Briefly, protein concentrations were determined with a BCA protein assay kit using BSA as the standard. Equal amounts of protein (100 μg) were fractionated by SDS‐PAGE and blotted to a PVDF membrane (Millipore, Bedford, MA). Membranes were blocked for 1 hour in using 5% nonfat milk in Tris‐buffered saline with Tween 20 (TBST), then probed overnight at 4°C with the following primary antibodies: BRCA1 (1:1000 dilution; Abcam, Cambridge, MA), FoxO3a and anti‐GAPDH (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CAQ), all in 5% milk TBST. After incubation with the primary antibodies, membranes were incubated with secondary antibody (1:8000 dilution; Alexa Fluor 700 goat anti‐mouse IgG [H+L] or Alexa Fluor® 800 goat anti‐rabbit IgG [H+L]; Invitrogen) in PBS at room temperature for 1 hour. Western blot bands were captured by using the Odyssey Infrared Imaging System (LI‐COR Biosciences, Lincoln, NE) and quantified with Odyssey software (v1.2; LI‐COR Biosciences) by measuring the band intensity (area×OD) in each group and normalizing to GAPDH as an internal control. Unless otherwise stated, western blot experiments were repeated 3 times.
Alexa fluor 700 goat anti mouse igg h l
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 700 goat anti‐mouse IgG [H+L] is a secondary antibody conjugated with the Alexa Fluor 700 fluorescent dye. It is designed to bind to the heavy and light chains of mouse immunoglobulin G (IgG) antibodies, allowing for their detection and visualization in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 800 goat anti rabbit igg h l, by Thermo Fisher Scientific (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab154769, by Abcam (2 mentions)
Las af, by Leica (2 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Sc 7292, by Abcam (2 mentions)
Hpx pl apo 63x 1.40 0 6 oil immersion objective, by Leica (2 mentions)
Tcs sp5 2 confocal microscope, by Leica (2 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (2 mentions)
Foxo3a, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Brca1, by Abcam (1 mentions)
Odyssey v1, by LI COR (1 mentions)
4 protocols using alexa fluor 700 goat anti mouse igg h l
1
Western Blot Analysis of BRCA1 and FoxO3a
2
Protein Expression Analysis by Western Blot
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Brie y, the protein concentrations were determined using a bicinchoninic acid protein assay kit and bovine serum albumin as the standard. Equal amounts of protein were fractionated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene uoride (PVDF) membrane (Millipore, Bedford, MA). The membranes were blocked for 2 h using 5% non-fat milk in Trisbuffered saline with Tween (TBST), and then probed overnight at 4 °C. Following incubation with the primary antibodies, the membranes were incubated with secondary antibody (1: 8000 dilution, Alexa Fluor® 700 goat anti-mouse IgG (H+L) or Alexa Fluor® 800 goat anti-rabbit IgG (H + L); Invitrogen) in PBS at room temperature for 1 h. The protein bands were imaged using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) and quanti ed using the Odyssey v1.2 software (LI-COR Biosciences, Lincoln, NE, USA) by measuring the band intensity (area × OD) in each group. The band intensities were normalized to that of GAPDH or tubulin as the internal control. The western blot experiments were repeated thrice unless otherwise stated. The antibodies used are listed in Additional le 1: Table S2.
3
Immunofluorescence Staining of Nuclear Proteins
4
Immunofluorescence Staining of Nuclear Proteins
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Immunofluorescence staining was performed at room temperature. Fixed cells were permeabilized with 0.1% triton (Sigma T8787) in 1× PBS for 10 minutes. Cells were blocked with 3% BSA (Sigma A7906) in 0.1% triton/PBS for 1 hour. Cells were incubated with primary antibody diluted in the blocking buffer for 2 hours [Lamin A/C (636) mouse mAb (1:800, SantaCruz sc-7292 lot C2219), Rad21 rabbit pAb (1:1000, abcam ab154769, lot GR3224138-10), CTCF rabbit pAb (1:800, Cell Signaling 2899, lot 2)]. Cells were washed with 0.1% triton/PBS 3 × 5 minutes. Cells were incubated with secondary antibodies [goat anti-rabbit IgG H&L Alexa Fluor 488 (1:1000, abcam ab15007, lot GR3225678-1), goat anti-mouse IgG H&L Alexa Fluor 700 (1:1000, invitrogen A-21036, lot 2084419)] diluted in the blocking buffer and conjugated tubulin antibody [anti-tubulin (YOL1/34)-AlexaFluor647, rat mAb, 1:100, abcam ab195884, lot GR281429-4] for 1 hour in the dark.
Cells were washed with 0.1% triton/PBS 1 × 5 minutes and then washed with 1× PBS 3 × 5 minutes. Coverslips were mounted to slides using ProLong Diamond Antifade Mountant with DAPI (Invitrogen P36962). For image acquisition, we used a Leica TCS SP5- II confocal microscope with 405 nm, 488 nm, 561 nm, and 633 nm lasers. Imaging we performed using a Leica HPX PL APO 63X/1.40-0.6 oil immersion objective with standard PMTs. Images were acquired using Leica LAS AF.
Cells were washed with 0.1% triton/PBS 1 × 5 minutes and then washed with 1× PBS 3 × 5 minutes. Coverslips were mounted to slides using ProLong Diamond Antifade Mountant with DAPI (Invitrogen P36962). For image acquisition, we used a Leica TCS SP5- II confocal microscope with 405 nm, 488 nm, 561 nm, and 633 nm lasers. Imaging we performed using a Leica HPX PL APO 63X/1.40-0.6 oil immersion objective with standard PMTs. Images were acquired using Leica LAS AF.
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