Diethyl nitrosamine, Proteo-Prep Immunoaffinity Albumin and IgG Depletion Kit, thiourea, dithiothreitol, iodine acetamide, sodium thiosulfate, and 2-dimensional gel electrophoresis standard protein were purchased from Sigma Company. Solid phase pH gradient dry strip (IPC strip pH 3-10 L, and 24 cm), interpublic buffer (pH : 3-10), ampholytes (pharmalyte, pH 3-10), low-molecular-weight protein, protein silver dye kits, urea, propylene phthalein amine, double propylene phthalein methylene amine, glycerin, glycine, nonidet P-40, 3-hydroxymethyl aminomethane, 3-[(3-bravery acyl amino propyl) dimethyl amine] propyl sulfonate, phenylmethylsulfonyl fluoride, Triton X-100, sodium dodecyl sulfate (SDS), EDTA, and filter column were purchased from Amershma Bioseiences Company. IPGhor isoelectric focusing (GE Healthcare), DALT-6 sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) electrophoresis apparatus, ImageScanner scanner, ImageMaster 2d Platinum 7.0 analysis software were purchased from GE Healthcare Company. ABI4800 tandem time-of-flight mass spectrometer (MALDI-TOF/TOF) is purchased from America ABI Co LTD. The primary and second antibodies of clusterin, NDRG2, and HSPa8 are purchased from Santa Cruz biotechnology Co, Ltd.
Ndrg2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
NDRG2 is a protein-coding gene that produces the N-Myc Downstream Regulated Gene 2 protein. This protein is involved in various cellular processes, including cell growth, differentiation, and stress response. The NDRG2 gene is expressed in different tissues and plays a role in the regulation of gene expression.
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Lab products found in correlation
Dithiothreitol (dtt), by Merck Group (1 mentions)
Sodium thiosulfate, by Merck Group (1 mentions)
Thiourea, by Merck Group (1 mentions)
Proteoprep immunoaffinity albumin and igg depletion kit, by Merck Group (1 mentions)
Iodine acetamide, by Merck Group (1 mentions)
Imagemaster 2d platinum 7.0 analysis software, by GE Healthcare (1 mentions)
Diethylnitrosamine, by Merck Group (1 mentions)
Hspa8, by Santa Cruz Biotechnology (1 mentions)
Rabbit polyclonal igg, by Abcam (1 mentions)
Mouse and rabbit specific hrp dab abc detection ihc kit, by Abcam (1 mentions)
α actin, by Agilent Technologies (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Pvdf blotting membrane, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Skim milk, by BD (1 mentions)
Ez capture mg, by ATTO Corporation (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
α actinin, by Santa Cruz Biotechnology (1 mentions)
4 protocols using ndrg2
1
2D Gel Proteomics of Diethyl Nitrosamine
2
Immunohistochemical Staining for ITLN1 and NDRG2
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Immunohistochemical staining was performed as previously described [20 (link),21 (link)], with antibodies specific for ITLN1 (Abcam, Cambridge, MA; Santa Cruz Biotechnology, Santa Cruz, CA; 1:200 dilutions) and NDRG2 (Santa Cruz Biotechnology; 1:200 dilution). The negative controls included parallel sections treated with omission of the primary antibody, in addition to an adjacent section of the same block in which the primary antibody was replaced by rabbit polyclonal IgG (Abcam Inc.) as an isotype control. The immunoreactivity in each tissue section was assessed by at least two pathologists without knowledge of the clinicopathological features of tumors. The degree of positivity was initially classified according to the percentage of positive tumor cells as the following: (−) < 5% cells positive, (1+) 6–25% cells positive, (2+) 26–50% cells positive, and (3+) >50% cells positive.
3
Histological Analysis of Calcified Aortic Valve
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Three consecutive sections from the AS valve analyzed by MALDI-IMS were used for the histological analysis. These sections were stained using Verhoeff-Van Giesson staining protocol for elastic fibers and oil red for lipids.
Besides the consecutive sections, we used 4 different AS valves, selecting 4 sections with increasing level of calcification of each one, for IHC analyses. In this case, sections were blocked with 10% BSA in PBS Buffer with 0.1% Tween 20 and incubated for 1 h with the primary antibodies at room temperature. Specifically, these analyses were performed with antibodies against CD68, α-actin (DAKO), NDRG2 and collagen α3 Type VI (Santa Cruz). Secondary antibody conjugated with biotin, streptavidin-peroxidase and DAB was used for all immunostainings (Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit, Abcam). Besides, additionally sections from 4 stenotic valves were used to study the distribution of NDRG2 and collagen α3 Type VI along the different stages of the lesion. For an impartial analysis of the DAB staining, an orthonormal transformation of the RGB images by using an ImageJ plugin based on Ruifrok and Johnston’s method for color deconvolution was performed37 (link).
Besides the consecutive sections, we used 4 different AS valves, selecting 4 sections with increasing level of calcification of each one, for IHC analyses. In this case, sections were blocked with 10% BSA in PBS Buffer with 0.1% Tween 20 and incubated for 1 h with the primary antibodies at room temperature. Specifically, these analyses were performed with antibodies against CD68, α-actin (DAKO), NDRG2 and collagen α3 Type VI (Santa Cruz). Secondary antibody conjugated with biotin, streptavidin-peroxidase and DAB was used for all immunostainings (Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit, Abcam). Besides, additionally sections from 4 stenotic valves were used to study the distribution of NDRG2 and collagen α3 Type VI along the different stages of the lesion. For an impartial analysis of the DAB staining, an orthonormal transformation of the RGB images by using an ImageJ plugin based on Ruifrok and Johnston’s method for color deconvolution was performed37 (link).
4
Western Blot Analysis of Cellular Proteins
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The cells were washed using DPBS, and the total protein was isolated by a protein extraction solution (iNtRON Biotechnology, Seongnam, Korea). The supernatant fractions were obtained by centrifugation at 12,000 rpm for 15 min at 4°C. The protein was mixed with a 5X sample buffer and separated on 9% or 12% SDS-polyacrylamide gel. Then, gel was transferred to a PVDF blotting membrane (GE healthcare Life Science, Wauwatosa, WI, USA), and the membrane was blocked with Tris-buffered saline plus 0.1% Tween-20 (TBS-T) containing 5% skim milk (BD Biosciences) or BSA (Gibco). The membrane was incubated with specific antibodies overnight at 4°C and washed by TBS-T. The antibodies against actin, α-actinin, NDRG2 and ICAM1 were purchased from Santa Cruz Biotechnology and the antibodies against NFATc1, MITF, STAT3, p-STAT3, JNK, p-JNK, ERK, p-ERK, p38, p-p38, AKT and p-AKT were obtained from Cell Signaling (Danvers, MA, USA). After attaching the secondary antibodies coupled to horseradish peroxidase, the blots of the membrane were visualized with an EZ-Western Lumi Plus solution (ATTO Corporation, Tokyo, Japan), pico EPD solution (ELPIS-Biotech., Inc, Daejeon, Korea) and Ez-Capture MG (ATTO Corporation).
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