Cell lysates were extracted using Triton X-100 lysing buffer, and lysate was quantified using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA, 23225). For each cell lysate, 20 μg of protein was run on a 4–15% SDS gel (Bio-Rad, Hercules, CA, USA, 4561083DC) and electrotransferred onto a PVDF membrane. The membrane was washed with TBS and blocked overnight with 3% BSA in TBST. The membrane was incubated with primary Rabbit anti-P-gp antibody (Abcam, Waltham, MA, USA, ab170904) for 2 h, washed 2× for 10 min with TBST, and incubated with a secondary Goat anti-Rabbit IgG HandL (Abcam, Waltham, MA, USA, ab97051) antibody for 1 h. Rabbit anti-β-actin antibody (Abcam, Waltham, MA, USA, ab8227) along with the MDR AT3B-1 cell line were used as controls (Figure 3 B). As per manufacturer’s instructions, the primary antibody detects the predominant protein band migrating in the region of 180–200 kDa and typically will demonstrate a smear on the membrane in the region of 150–300 kDa due to the glycosylation profile of the protein [66 (link)].
Goat anti rabbit igg h and l
Manufactured by Abcam
Sourced in United States
Goat anti-rabbit IgG H and L is a secondary antibody that binds to the heavy and light chains of rabbit immunoglobulin G (IgG). It is typically used in immunoassays and other applications that require the detection of rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Abcam (4 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (3 mentions)
Thunder imager 3d cell culture microscope, by Leica (3 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sds gel, by Bio-Rad (1 mentions)
Rabbit anti β actin antibody, by Abcam (1 mentions)
Tcs sp5 microscope, by Leica (1 mentions)
Alexa fluor 647, by Abcam (1 mentions)
Anti muc1 c, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
5 protocols using goat anti rabbit igg h and l
1
Western Blot Analysis of P-glycoprotein
2
Immunofluorescence Staining of BT-549 Cells
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BT-549 cells were stained as described.9 (link) Briefly, cells were fixed in 4% paraformaldehyde and were incubated with 0.1% Triton X-100 at room temperature for 10 min. The samples were blocked with 3% Normal Goat Serum (Gibco), incubated with anti-MICA (PA5-35346, 1:20 dilution; Thermo Fisher Scientific) or anti-MICB (NBP2-56506, 0.9 μg/mL; Novus Biologicals, Centennial, Colorado, USA) at 4°C overnight and incubated with goat antirabbit IgG H and L labeled with Alexa Fluor 488 (Abcam). Invitrogen ProLong Gold Antifade Mountant with DAPI (Invitrogen) was used to stain the nuclei. The cells were analyzed by a Leica THUNDER Imager 3D Cell Culture microscope as described.9 (link)
3
Immunofluorescent Analysis of DNA Damage in Mammospheres
4
Immunofluorescent Analysis of Mammospheres
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Mammospheres were fixed with 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 15 minutes. Samples were incubated with 1% Triton X-100 (Sigma-Aldrich) at room temperature for 10 minutes and blocked with 5% normal goat serum (Gibco). The mammospheres were attached to slides via cytospin at low speed (Shandon Cytospin 3; Shandon Scientific) and stained with anti-γH2AX (#9718, 1:400 dilution, CST) and goat anti-rabbit IgG H and L labeled with Alexa Fluor 488 (Abcam) as described (41 (link)). Nuclei were stained with ProLong Gold Antifade Mountant with DAPI (Invitrogen). Cells were imaged using a Leica THUNDER Imager 3D Cell Culture microscope, as described (41 (link)).
5
Immunofluorescence Analysis of MUC1 and MYC in Cancer Cells
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HPAF-II and AsPC-1 cells were fixed in 3.7% paraformaldehyde (Sigma) at room temperature for 15 min. These samples were washed three times with phosphate-buffered saline and then incubated with 0.3% Triton X-100 (Sigma) at room temperature for 10 min. The samples were blocked with 3% BSA and incubated with anti-MUC1-C (#MA5-11202, 1:50 dilution; Thermo Fisher Scientific) or anti-MYC (#5605, 1:500 dilution; CST) at 4°C overnight. The samples were then incubated with goat anti-Armenian hamster IgG H and L labeled with Alexa Fluor 488 (Abcam) and goat anti-rabbit IgG H and L labeled with Alexa Fluor 647 (Abcam) at room temperature for 1 h. DAPI (Sigma) was used for staining of nuclei. The cells were analyzed by confocal microscopy using an inverted Leica TCS SP5 microscope. Immunofluorescence intensities were calculated using ImageJ software.
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