Total protein was extracted using RIPA lysis buffer (Thermo Scientific, United Kingdom), and its concentration was determined with an enhanced bicinchoninic acid assay kit (CWBio, China). Around 40 mg protein per sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (CWBio, China). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight with primary antibodies targeting MRTO4 (1:2000, ab212044, Abcam, United States), BOP1 (1:3000, ab32053, Abcam, United States), PES1 (1:5000, ab56701, Abcam, United States), NDUFS8 (1:3000, ab226760, Abcam, United States), NDUFS6 (1:3000, ab226760, Abcam, United States), NDUFA8 (1:3000, ab226760, Abcam, United States) and β-actin (1:5000, ab8226, Abcam, United States) at 4 °C. The membranes were washed thrice with TBST (Tris-buffered saline with Tween 60), and probed with horseradish peroxidase-conjugated secondary antibody (1:5000) for 1.5 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence system, and the membranes were exposed to X-ray films (Bio-Rad, United States). Densitometric analysis was performed using Image Pro-Plus software (Media Cybernetics, United States), and relative protein expression levels were normalized to β-actin.
Ab212044
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-PCNA antibody [PC10] (ab212044) is a mouse monoclonal antibody that recognizes the Proliferating Cell Nuclear Antigen (PCNA) protein. PCNA is a cofactor of DNA polymerase delta and is involved in DNA replication and repair.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Ab56701, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Ab32053, by Abcam (2 mentions)
Enhanced bicinchoninic acid assay kit, by CWBIO (2 mentions)
Polyvinylidene difluoride membranes, by CWBIO (2 mentions)
Ab226760, by Abcam (2 mentions)
X ray film, by Bio-Rad (2 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Ab52894, by Abcam (1 mentions)
Ab32430, by Abcam (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Ab8245, by Abcam (1 mentions)
Image software, by Media Cybernetics (1 mentions)
4 protocols using ab212044
1
Western Blot Analysis of Mitochondrial Proteins
2
BEAS-2B Cell Protein Expression Analysis
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BEAS-2B cells were lysed with RIPA buffer, with the total protein extracted. The protein per lane was loaded onto a 10% SDS-PAGE and transferred to the PVDF membranes (GE Healthcare, USA). The membranes were probed with primary antibodies against EGFR (ab52894, Abcam, Cambridge, UK), p-EGFR (ab32430, Abcam), HSPA6 (ab212044, Abcam), and GAPDH (ab8245, Abcam) overnight at 4°C. Then, the membranes were rinsed three times and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, and HRP substrates were added to visualize the protein bands. The housekeeping gene GAPDH was used as a control for normalization. Densitometric analysis of immunoblots was performed using ImageJ software (USA).
3
Immunohistochemical Analysis of HSPA6 Expression
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Tissue specimens used in this study were fixed, dehydrated, embedded and then cut into 5 μm‐thick sections. IHC was performed using a kit as per the manufacturer's protocol. Following antigen retrieval in hot citrate buffer, those sections were incubated the whole night with anti‐HSPA6 primary antibody (1:200, ab212044, Abcam) at 4°C. Each section of those was washed three times with TBST for 5 min, and incubated with a rabbit secondary antibody at RT for 1 h. The staining intensity of HSPA6 and the percentage of positively stained cells in tissues were scored by three independent pathologists in a blinded fashion. Based on the percentage of positively stained cells, the samples were graded as 4 (>76%), 3 (51%–75%), 2 (26%–50%), 1 (6%–25%) and 0 (≤5%). The staining intensity was graded as strong, 3 moderate, 2 weak 1 and negative (0). The total scores were calculated by multiplying the staining intensity score with that of the percentage of positive cells and ranged from 0 to 12.16 Subsequently, the samples were stratified as HSPA6high and HSPA6low based on scores ranging from 6 to 12 and 0 to 4, respectively.
4
Western Blot Analysis of Stress Proteins
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Total protein extracted by RIPA lysis buffer (Thermo Scientific) was determined with an enhanced bicinchoninic acid assay kit (CWBio). The protein from each sample was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (CWBio). The membranes blocked with 5% non‐fat milk for 1 h at room temperature (RT) later were incubated the whole night with primary antibodies targeting HSPA6 (1:2000, ab212044, Abcam), Cyclin B1 (1:3000, ab32053, Abcam), YAP1 (1:5000, ab56701, Abcam), p‐S127‐YAP1 (1:3000, ab226760, Abcam) and β‐actin (1:5000, ab8226, Abcam) at 4°C. The membranes were washed three times with tris‐buffered saline with Tween 60 (TBST) and probed with horseradish peroxidase‐conjugated secondary antibody (1:5000) for 1.5 h at RT. Protein bands were exposed by a chemiluminescence system, and the membranes were visualized by X‐ray films (Bio‐Rad). Densitometric analysis was performed by image software (Media Cybernetics). The relative protein expression levels of proteins measured in this study were normalized to β‐actin.
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