Radio-Immunoprecipitation lysis (BB-3209, BestBio, Shanghai, China) was used to isolate the total protein from the cells. The protein was separated by SDS-PAGE and transferred onto the polyvinylidene fluoride membrane. The membrane was allowed to probe with primary antibodies including rabbit monoclonal antibodies to proliferating cell nuclear antigen (PCNA) (1:1,000, ab92552, Abcam Inc., Cambridge, UK), Ki-67 (1:5,000, ab92742, Abcam Inc.), matrix metalloproteinase (MMP)-2 (1:1,000, ab92536, Abcam Inc.), MMP-9 (1:1,000, ab38898, Abcam Inc.), Cyclin D1 (1 : 200, ab16663, Abcam Inc.), and Cyclin-dependent kinase 4 (CDK4) (1:1,000, ab108357, Abcam Inc.). Horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:1,000, Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) was added and incubated with the membrane. Target protein relative expression = gray value of the target protein band/gray value of GAPDH.
Ab92552
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab92552 is a rabbit monoclonal antibody against the protein Histone H3. It is intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab16667, by Abcam (27 mentions)
Ab32503, by Abcam (20 mentions)
Ab181602, by Abcam (19 mentions)
Pvdf membrane, by Merck Group (19 mentions)
Ab205718, by Abcam (18 mentions)
Ab32124, by Abcam (15 mentions)
Ab76003, by Abcam (12 mentions)
Ab8226, by Abcam (12 mentions)
Ab92536, by Abcam (11 mentions)
Ab16663, by Abcam (10 mentions)
Ab6721, by Abcam (10 mentions)
Ab9485, by Abcam (10 mentions)
Ab15580, by Abcam (9 mentions)
Ab182858, by Abcam (9 mentions)
Bca protein assay kit, by Beyotime (9 mentions)
Ab2302, by Abcam (9 mentions)
Ab92742, by Abcam (8 mentions)
Ab8227, by Abcam (8 mentions)
Ripa lysis buffer, by Beyotime (7 mentions)
Ab40772, by Abcam (7 mentions)
171 protocols using ab92552
1
Western Blot Analysis of Cell Signaling Proteins
2
Xenograft and Liver Metastasis Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Xenograft experiments were performed with the approval of the Institutional Animal Care and Use Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Five-week-old male athymic BALB/c nude mice were used. A total of 2 × 106 indicated cells were subcutaneously inoculated into the flanks of mice. Subcutaneous xenograft volumes were measured every 7 days using a calliper and calculated by the formula V = 0.5 × a × b2 (a, the long diameter; b, the short diameter). On the 21st day after inoculation, subcutaneous xenografts were resected and weighed. Subcutaneous xenografts were subjected to immunohistochemistry (IHC) staining using the primary antibodies against Ki67 (ab15580, 1:200, Abcam, Cambridge, MA, USA), PCNA (ab92552, 1:200, Abcam), or cleaved caspase-3 (#9661, 1;200, Cell Signalling Technology). Furthermore, subcutaneous xenografts were also subjected to TUNEL assays using the One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology) following the provided instruction. A total of 2 × 106 indicated cells were intrasplenically injected into the mice to construct liver metastasis model. On the 35th day after injection, the livers were resected and subjected to haematoxylin–eosin (H&E) staining.
3
Protein Expression Analysis in Tissue Homogenate
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBS was used to wash fresh tissue which was grounded into a homogenate, and then RIPA buffer was used to lyse them to obtain total protein. Protein concentrations were determined by the BCA assay (Solarbio, PC0020, China). Samples (30 μg protein) were resolved on 8% SDS-PAGE gel and transferred to PVDF at 4°C. Membranes were blocked in fat-free milk combined with TBST for 1 hour at 25°C and immunoblotted with the appropriate diluted primary antibody KIF20A (Abcam, ab104118, UK; 1 : 1000 dilution for Western blot and 1 : 200 dilution for IHC-P), PCNA (Abcam, ab92552, UK; 1 : 1000 dilution for Western blot), Ki67 (Abcam, ab16667, UK; 1 : 1000 dilution for Western blot), MMP2 (Abcam, ab37150, UK; 1 : 500 dilution for Western blot), MMP9 (Abcam, ab76003, UK; 1 : 5000 dilution for Western blot), and GAPDH (SunGene Biotech, KM9002, China; 1 : 5000 dilution for Western blot) at 4°C overnight. Then, membranes were washed 10 minutes three times and incubated with the secondary antibody for 1 hour at 25°C. Finally, the blots were visualized by Imager.
4
Western Blot Analysis of Cell Cycle Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed by Cell LyticäM (Sigma) and added to gel-loading buffer. After boiling for 10 min, proteins were resolved by SDS–polyacrylamide gel electrophoresis and electroblotted onto a polyvinylidene fluoride membrane (Millipore Co., Billerica, MA, USA). Membranes were blocked with 3% BSA and incubated with indicated antibodies. After washing in PBS containing 0.5% Tween-20, the bound primary antibody was detected with anti-mouse IgG or anti-rabbit IgG (Zhongshan Co., Beijing, China). Following washes, the antibody-bound protein was visualised by enhanced chemiluminescence. Antibodies used are as follows: anti-cyclin A (BS1084, Bioworld Technology, St Louis Park, MN, USA); anti-cyclin B1 (ab32053, Abcam, Cambridge, MA, USA); anti-cyclin D1 (2261-1, Epitomics, Burlingame, CA, USA); anti-PCNA (ab92552, Abcam); mouse-anti-HA monoclonal antibody (TA-04, Zhongshanjinqiao, Beijing, China); rabbit anti-HA polyclonal antibody (51064-2-AP, Proteintech, Chicago, IL, USA); anti-αvβ3 (CBL544, Millipore); and anti-β-Actin (TA-09, Zhongshanjinqiao).
5
Protein Expression Analysis of Bronchial Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bronchial tissue samples were lysed in 1 mL of cell extraction buffer. Lysates were incubated in radio-immunoprecipitation assay buffer and kept at 4°C, for 2 h, followed by centrifugation at 12,000×g for 30 min at 4°C, and the supernatants collected. Total proteins were separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis on 8% gels, transferred onto polyvinylidene fluoride membranes, and blocked in TBST (plus 5% skim milk powder, pH 7.4, tris-buffered saline [TBS] with 0.24% Tween-20). Membranes were then incubated overnight at 4°C with the following primary antibodies: p-PI3K (Abcam, ab86714, 1:1,000), p-PKB (Abcam, ab81283, 1:5,000), p-PCNA (Abcam, ab92552, 1:3,000), p-AR (Abcam, ab3442, 1:1,000), and β-actin (Abcam, ab8226, 1:2,000). Blots were washed three-times for 15 min in TBST and treated with an horseradish peroxidase-conjugated polyclonal goat anti-rabbit antibody (Beyotime, A0208, 1:1,000) for 2 h at room temperature. Membranes were washed three-times for 10 min, and HRP substrate used to detect the HRP-secondary antibody bound to the proteins of interest, based on the manufacturer’s instructions. The p-PI3K, p-PKB, p-PCNA, and p-AR protein expression levels were standardized to those of β-actin.
6
Quantification of Stem Cell Markers in Rat Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The colon, liver, spleen, kidney and lung tissues of the rats (n = 5) were fixed in 4% paraformaldehyde overnight and then dehydrated in 30% sucrose solution. The tissues were embedded in optimal cutting temperature (OCT) compound and cut into 5-μm-thick frozen sections. After soaking with PBS, the sections were placed in 5% normal goat serum (abs933, Absin, Shanghai, China) and incubated with anti-Bmi1 (1:200, ab14389, Abcam), anti-Musashi1 (1:200, c-135,721, Santa Cruz Biotechnology), anti-Sox9 (1:100, ab3697, Abcam) and anti-PCNA antibodies (1:200, ab92552, Abcam) at 4 °C overnight. Next, the sections were washed in PBS and incubated at 37 °C for 1 h with anti-mouse IgG (1:500, 8890, Cell Signaling Technology) and anti-rabbit IgG (1:500, 8889, Cell Signaling Technology). Sections were then stained with DAPI and anti-fading medium before observation by a laser scanning confocal microscope (LSM 510 META; Zeiss, Germany), and the results were semi-quantitatively analysed with ImageJ (National Institutes of Health, Bethesda, USA).
7
Western Blot Analysis of CAPS1 and PCNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression of CAPS1 and PCNA was detected by Western blotting, as previously described [24 (link)]. Briefly, cells were lysed using radioimmunoprecipitation assay (RIPA) buffer and subjected to immunoblotting using anti-CAPS1 (PRS4561; Sigma-Aldrich, St. Louis, MO, USA), anti-PCNA (AB92552; Abcam, Cambridge, UK), and anti-GAPDH (AB181603; Abcam) antibodies. Proteins were visualized using an enhanced chemiluminescence kit (Pierce, Brookline, MA, USA).
8
Quantitative Western Blot Analysis of Apoptosis and Angiogenesis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TP was extracted from pancreatic tissue using RIPA buffer and a protease inhibitor (Beyotime, China), and the protein concentration was determined by the BCA method. The protein samples were denatured in a metal bath at 95 ℃, separated by SDS-PAGE and transferred onto PVDF membranes. The samples were subsequently blocked with 5% nonfat milk in PBS for 1 h and then incubated with the following primary antibodies overnight at 4 ℃: Rabbit-derived Bcl2 (ab182858; Abcam), Bcl-xL (ab32370; Abcam), proliferating cell nuclear antigen (PCNA) (ab92552; Abcam), and vascular endothelial growth factor (VEGF) (ab32152; Abcam). The membranes were then incubated with goat anti-rabbit HRP-conjugated secondary antibodies at room temperature for 2 h. GAPDH was used as the internal control. Immunoreactions were detected using enhanced an chemiluminescence reagent and an Amersham Imager 600 system (GE Healthcare Life Sciences), and the grayscale values of the bands representing the targeted proteins were quantitated using ImageJ software.
9
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted using RIPA lysis buffer (Beyotime) and protein concentration was determined using BCA Protein Assay Kit (Beyotime). Equal amounts of protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Nonspecific binding proteins were blocked with 5% non-fat milk for 1.5 h at room temperature. Subsequently, membranes were incubated overnight at 4̊C with antibodies against NCOA5 (Bioworld, BS67243, 1:1,000), Ki67 (Abcam, ab16667, 1:1,000), PCNA (Abcam, ab92552, 1:1,000), MMP2 (Abcam, ab92536, 1:1,000), MMP9 (Abcam, ab76003, 1:1,000), VEGFA (Thermo Fisher Scientific, OPA1-10110, 1:200), VEGFR2 (Abcam, ab134191, 1:1,000), TPX2 (Abcam, ab252945, 1:1,000) and GAPDH (Abcam, ab9485, 1:2,500). On the next day, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, ab6721, 1:3,000) for 2 h at room temperature. GAPDH served as the endogenous control. Enhanced chemiluminescence (ECL) kit was applied to develop the protein bands and the blots were visualized and analyzed by a Bio-Rad imaging system (Bio-Rad).
10
Quantification of Nrf2 and β-TrCP Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein and nucleoprotein (for Nrf2 and β-TrCP) in the ipsilateral peri-infarct hemisphere were extracted, and the protein concentration was determined by bicinchoninic acid protein (BCA) assay. The protein samples were then denatured by boiling for 10 min. Then, 20 μg of each protein sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V for 100 min. After transfer to polyvinylidene fluoride (PVDF) membrane, the separated protein bands were incubated with individual primary antibodies (caspase-3 1:500, ab197202, Abcam; AKT, 1:1000, Cat# 9272, CST; p-AKT, 1:1000, Cat#9271, CST; GSK-3β, 1:1000, Cat# 9315, CST; p-GSK-3β, 1:1000, Cat# 9336, CST; β-TrCP, 1:1000, Cat# 4394, CST; Nrf2 1:1000, ab92946, Abcam; PCNA, 1:1000, ab92552, Abcam; HO-1, 1:2000, ab52947, Abcam; NQO1, 1:10000, ab80588, Abcam) at 4 °C overnight, followed by incubation with corresponding secondary antibodies at room temperature for 2 h. The developed bands were observed under a gel imaging system.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!