Ab227958

Manufactured by Abcam

Sourced in United States

Ab227958 is a lab equipment product offered by Abcam. It is a tool designed for use in research and laboratory applications, but a detailed description of its core function cannot be provided in a concise, unbiased, and factual manner without risk of extrapolation. Therefore, the description for this product is not available.

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Lab products found in correlation

Ab32572, by Abcam (2 mentions) Two step ihc kit, by ZSGB-BIO (1 mentions) Strataquest software, by TissueGnostics (1 mentions) Cervical cancer tissue microarray, by Shanghai Outdo Biotech Co. (1 mentions) K8002, by Agilent Technologies (1 mentions) Ab179445, by Abcam (1 mentions) Ab177467, by Abcam (1 mentions) Ab181602, by Abcam (1 mentions) Mg132, by MedChemExpress (1 mentions) Ab97051, by Abcam (1 mentions) Pvdf membrane, by Merck Group (1 mentions)

6 protocols using ab227958

Quantitative IHC Analysis of NEK2 in ccRCC

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Immunohistochemical (IHC) analysis was conducted using tissue microarray (TMA) of ccRCC and adjacent normal tissue specimens according to the protocol of the Two-Step IHC Kit (ZSGB-BIO, Beijing, China) using an anti-NEK2 antibody (ab227958, 1:200; Abcam, Cambridge, UK). The specimens of TMA were reviewed by one uropathologist and the most representative areas of renal tumor cells and adjacent tumor stroma were selected for the donor block. The TMAs were made by using a tissue-arraying instrument (Manual Tissue Microarrayer Quick-Ray ®). Cores with 2 mm diameter from per-donor block were diverted into a recipient block microarray.
The StrataQuest software (TissueGnostics, Vienna, Austria) was used to scan and analyze the images to quantify the tumor tissues, para-carcinoma tissues and NEK2 positive cells in the TMA. We multiplied the quantitative value of positive staining intensity (negative = 1, weak = 2, moderate = 3, strong = 4) by percentages of positive staining (0% = 0, ≤25% = 1, 26%–50% = 2, 51%–75% = 3, ≥76% = 4), and the data obtained was weighted and set into four levels (Ⅰ, Ⅱ, Ⅲ, Ⅳ). All results were verified by three experienced independent investigators.

Wang C., Huang Y., Ma X., Wang B, & Zhang X. (2021). Overexpression of NEK2 is correlated with poor prognosis in human clear cell renal cell carcinoma. International Journal of Immunopathology and Pharmacology, 35, 20587384211065893.

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Immunohistochemical Analysis of NEK2 in Cervical Cancer

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A cervical cancer tissue microarray, which contained 41 cervical carcinoma tissues and paired paracarcinoma tissues, was obtained from Shanghai Outdo Biotech (Shanghai, China). IHC analysis was performed as previously described [32 (link)–34 (link)]. Briefly, the tissue sections were deparaffinized, rehydrated, and blocked with goat serum. After incubation with the anti-NEK2 antibody (1:100, ab227958, Abcam) overnight at 4 °C, the sections were washed three times with PBS and incubated with an HRP-conjugated secondary antibody (1:300, K8002, Dako) for 30 min at room temperature. Following three 5-min rinses in PBS, the stained sections were reacted with 3,3′-diaminobenzidine for 10 min and then counterstained with 0.1% hematoxylin. The staining in tumor and normal tissues was scored, and the staining percentage was determined. The score calculated by multiplying the values assigned to the staining intensity and percentage was used to evaluate the expression of NEK2.

Xu T., Zeng Y., Shi L., Yang Q., Chen Y., Wu G., Li G, & Xu S. (2020). Targeting NEK2 impairs oncogenesis and radioresistance via inhibiting the Wnt1/β-catenin signaling pathway in cervical cancer. Journal of Experimental & Clinical Cancer Research : CR, 39, 183.

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Antibody-based Protein Analysis

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An anti-NEK2 antibody (ab227958, Abcam, USA), an anti-GAPDH antibody (ab181602, Abcam, USA), a recombinant anti-E2F1 antibody (ab179445, Abcam, USA), and an anti-IGF2 antibody (ab177467, Abcam, USA) were used in the current study. The related reagents included the proteasome inhibitor MG132 (Z-Leu-Leu-Leu-al) (MedChemExpress, USA) and the protein synthesis inhibitor cycloheximide (CHX) (Abcam, USA).

Gu S., Yasen Y., Wang M., Huang B., Zhou Y, & Wang W. (2024). NEK2 promotes the migration, invasion, proliferation of ESCC and mediates ESCC immunotherapy. Heliyon, 10(9), e29682.

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Immunohistochemical Analysis of NEK2 Protein

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Immunohistochemistry was performed as previously described [31 (link)]. Anti-NEK2 primary antibody was purchased from Abcam (ab227958). Goat antirabbit IgG purchased from Abcam (ab97051) was used as the secondary antibody. Tissues embedded with paraffin were cut into 4-mm sections followed by deparaffinized, rehydrated, and stained with primary antibodies overnight at 4 °C. Afterward, the slides were incubated with corresponding secondary antibodies and stained with DAB. Tissues embedded with paraffin were cut into 4-mm sections. The sections were deparaffinized, rehydrated, and stained with primary antibodies overnight at 4 °C. The slides were subsequently incubated with secondary antibodies and stained with DAB. At last, hematoxylin was used to counterstain the slides and images were taken under the light microscope.

Xiang J., Alafate W., Wu W., Wang Y., Li X., Xie W., Bai X., Li R., Wang M, & Wang J. (2022). NEK2 enhances malignancies of glioblastoma via NIK/NF-κB pathway. Cell Death & Disease, 13(1), 58.

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Protein Extraction and Western Blot Analysis

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The total protein was extracted from each sample using RIPA buffer containing protease inhibitors. Total cell lysates containing 10 μg of protein were electrophoresed on a SDS-PAGE and then transferred to PVDF membranes (Millipore, MA, USA). The sample was blocked with 5% fat-free milk for 1 h, and incubated with primary antibodies for 1 h and with secondary antibodies for 1 h. The primary antibodies, including anti-Nek2B (ab227958; Abcam) at 1:2000 and anti-β-catenin (ab32572; Abcam) at 1:5000 were used in the present study. The blots were visualized using ECL assay.

Shen H., Yan W., Yuan J., Wang Z, & Wang C. (2019). Nek2B activates the wnt pathway and promotes triple-negative breast cancer chemothezrapy-resistance by stabilizing β-catenin. Journal of Experimental & Clinical Cancer Research : CR, 38, 243.

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Immunohistochemical Analysis of Nek2B and β-Catenin

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Immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded sections of surgical specimen as described previously [15 (link)]. The primary antibody rabbit monoclonal anti-human Nek2B antibody (ab227958, Abcam, Cambridge, MA, USA) at 1:250 dilution or mouse monoclonal anti-human β-catenin antibody (ab32572, Abcam) at 1:200 dilution was incubated overnight at 4 °C. For scoring Nek2B and β-catenin expression, the intensity (intensity score: 0 = negative, 1 = weak, 2 = moderate, and 3 = strong) and percentage of the total cell population (proportion score: 0, 0%; 1, <25%; 2, 25–49%; 3, 50–74%; 4, ≥ 75%) were evaluated for each case. The expression was regarded as negative (intensity multiply proportion scores 0–1) or positive (intensity multiply proportion scores ≥2) using high-powered (× 200) microscopy.

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