For staining the following antibodies and dilutions were used: for TAP1, rabbit polyclonal H-300 (1:100); for TAP2, rabbit polyclonal H210 (1:100), both from Santa Cruz Biotechnology, Inc (Dallas, TX, USA); for LMP2, rabbit polyclonal antibody ab3328 (1:1000); for LMP7, ab3329 (1:500), both from Abcam (Cambridge, United Kingdom). As secondary antibodies, BA-1000 anti-rabbit (1:200) and BA-2000 anti-mouse (1:200) both from Vector Laboratories (Burlingame, CA, USA) were used. The tumors included in the present study were formerly evaluated for LMP10 with ab C-2 (Santa Cruz Biotechnology, Inc) [27] (link), and for HLA class I with mouse monoclonal antibodies HCA-2 and HC-10 [15] (link), [16] (link). Data from these studies have been included in the present study for comparison to other APM components.
H 210
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The H-210 is a laboratory centrifuge designed for general-purpose applications. It is capable of separating and isolating various biological samples, such as cells, organelles, and macromolecules. The H-210 features a versatile rotor system and can accommodate a range of sample volumes and tube sizes.
Automatically generated - may contain errors
Lab products found in correlation
Ab3328, by Abcam (1 mentions)
Ab3329, by Abcam (1 mentions)
H 300, by Santa Cruz Biotechnology (1 mentions)
Phenylmethanesulfonyl fluoride, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Na9340, by GE Healthcare (1 mentions)
Na931, by GE Healthcare (1 mentions)
Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Nupage mops buffer, by Thermo Fisher Scientific (1 mentions)
Rad21, by Santa Cruz Biotechnology (1 mentions)
Smc1a, by Fortis Life Sciences (1 mentions)
Stag2, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ae1 ae3, by Agilent Technologies (1 mentions)
K005 ap red detection system, by Agilent Technologies (1 mentions)
Mib 1, by Agilent Technologies (1 mentions)
Vim3b4, by Agilent Technologies (1 mentions)
4 protocols using h 210
1
Immunohistochemical Analysis of APM Components
2
Western Blot Analysis of Brachyury
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Protein lysates from H460, PANC-1 and chordoma cells were prepared with RIPA buffer (Cell Signaling Technology) supplemented with 1 mM Phenylmethanesulfonyl fluoride (Sigma-Aldrich); five to 10 μg of protein was run in each lane. The following primary antibodies against brachyury were used: monoclonal rabbit (MAb 54-1, 1 μg/ml), monoclonal murine ab57480 (purchased from Abcam, 1 μg/ml), and polyclonal rabbit H-210 (purchased from Santa Cruz Biotechnology, Inc; 1/200 dilution). IRDye-800CW conjugated goat anti-mouse or anti-rabbit secondary antibodies (LI-COR Biosciences) were utilized at a 1:5000 dilution. Hybridoma supernatants were screened at a 1:500 dilution. All western blots were imaged and quantified using the Odyssey Infrared imaging system (LI-COR Biosciences).
3
Analyzing Cohesin Complex Protein Levels
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Protein was extracted from equal cell numbers in a high urea buffer (48% urea, 15 mM Tris-HCl, pH 7.5, 8.7% glycerin, 1% SDS, 0.004% bromophenol blue, and 143 mM β-mercaptoethanol). Protein levels were measured using the Bradford assay. Equal amounts of protein were loaded on NuPAGE Novex 4–12% Bis-Tris protein gel (NP0321; Life Technologies) and run with NuPAGE MOPS buffer (NP0001; Life Technologies). Proteins were transferred to Immobilon P (IPVH00010; EMD Millipore). Membrane was blocked in 5% nonfat milk in TBST and incubated with antibodies for Rad21 (H-210; Santa Cruz Biotechnology, Inc.), Smc1a (A300-055A; Bethyl Laboratories, Inc.), Smc3 (ab9263; Abcam), Stag2 (J-12; Santa Cruz Biotechnology, Inc.) and Actin B (C4; EMD Millipore). Secondary antibodies used were HRP conjugated (NA9340 and NA931; GE Healthcare). Blot was visualized using ECL (PI34077 and PI34095; Thermo Fisher Scientific).
4
Immunohistochemistry of Chordoma Cells
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Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissue via the avidin-biotin-complex-method using the K005 AP/RED Detection System (Dako, Glostrup, Denmark). The following antibodies were used: monoclonal antibodies against brachyury (H-210, Santa Cruz, Dallas, USA, 1:100), epithelial membrane antigen (EMA; E29, Dako, 1:500), vimentin (VIM3B4, Dako, 1:300), cytokeratin (AE1 + AE3, Dako, 1:100), Ki-67 (MIB-1, Dako, 1:200), p53 (DO-7, Dako, 1:500) and the polyclonal antibodies against S100-protein (Dako, 1:1000) and HOXA10 (OriGene Technologies, Rockville, USA, 1:100). Appropriate positive and negative controls were included.
The ratio of positive chordoma cells was characterized as follows: “no immunoreactivity detected” (−), “immunoreactivity in up to 30% (+), “immunoreactivity in more than 30% and up to 70%” (++) and “immunoreactivity in more than 70%” (+++) of the total number of chordoma cells in the section53 (link).
The ratio of positive chordoma cells was characterized as follows: “no immunoreactivity detected” (−), “immunoreactivity in up to 30% (+), “immunoreactivity in more than 30% and up to 70%” (++) and “immunoreactivity in more than 70%” (+++) of the total number of chordoma cells in the section53 (link).
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