Proteins were isolated with RIPA buffer (JRDUN Biotech., Shanghai, China) that contained protease and phosphatase inhibitors, which then went through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA), and blocked by 5% nonfat milk. The membranes were probed overnight at 4°C with antibodies against: NEDD4L (Ab240753, Abcam, Cambridge, MA, USA), p21 (Ab107099, Abcam), p27 (Ab32034, Abcam), ENO1 (Ab227978, Abcam) and GAPDH (#5174, Cell Signaling Technology). After wash, membranes were treated with horseradish peroxidase conjugated secondary antibody for 1 h, then with substrate (ECL; Bio-Rad, Richmond, CA, USA) followed by signal reading.
Ab107099
Manufactured by Abcam
Sourced in United States
Ab107099 is a recombinant antibody that recognizes the KRAS protein. The antibody is produced in Escherichia coli and is supplied as a purified protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab240753, by Abcam (1 mentions)
Ab227978, by Abcam (1 mentions)
Ab32034, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Donkey anti rat af594, by Thermo Fisher Scientific (1 mentions)
Af594 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Donkey anti goat af488, by Thermo Fisher Scientific (1 mentions)
Click it edu alexa fluor 594 imaging kit, by Thermo Fisher Scientific (1 mentions)
Mec13, by BD (1 mentions)
Bs 4056r, by Bioss Antibodies (1 mentions)
Dab substrate, by ZSGB-BIO (1 mentions)
Zli 9019, by ZSGB-BIO (1 mentions)
Ab189034, by Abcam (1 mentions)
Ab32129, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
A0208, by Beyotime (1 mentions)
Ab54210, by Abcam (1 mentions)
5 protocols using ab107099
1
Western Blot Analysis of Cell Signaling
2
Multimodal Immunohistochemistry Analysis
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Immunohistochemistry was performed as described previously (Richardson et al., 2012 ). 10 μm sections were used for all studies. Primary antibodies used: rat ant‐p21 (HUGO291, Abcam ab107099), goat‐anti‐troponin C (Abcam, ab30807), rabbit anti‐p16 (Rockland, 100‐401‐170) and rat anti‐CD31 (MEC13.3, BD Biosciences, 550274). Secondary antibodies used were donkey anti‐rat AF594 (Life Technologies, A21209), donkey anti‐goat AF 488 nm (Life Technologies, A11055), donkey anti‐rabbit AF594 (Life Technologies, R37119), donkey anti‐goat AF488 (Life Technologies, A11055) and donkey anti‐mouse AF647 (A31571, Life Technologies). Slides were mounted in Vectarshield containing DAPI (Sigma, MBD0015). 5‐ethynyl‐2‐deoxyuridine (EdU) labeling performed with Invitrogen Click‐iT EdU Alexa Fluor 594 Imaging Kit (Life Technologies, C10339).
3
Immunohistochemical Analysis of Tumor Tissues
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For immunohistochemical (IHC) staining, formaldehyde-fixed, paraffin-embedded sections of tumor tissues were obtained from mouse models. Briefly, the deparaffinized sections were then treated with methanol containing 3% hydrogen peroxide for 15 min. The sections were washed with PBS and blocked with blocking serum for 30 min at room temperature. The sections were then incubated with primary antibodies, including anti-SMURF2 (bs-4056R, Bioss), anti-ID2 (MA5-32891, Invitrogen), and anti-p21 (ab107099, Abcam) at 4°C overnight. The following day, the primary antibodies were washed off and sections were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The nuclei were stained with hematoxylin, and DAB substrate (ZLI-9019, ZSGB-Bio, Beijing, China) was added to detect the proteins. The obtained immunohistochemistry images were analyzed with following scoring criteria. Cells with 0% staining were scored as 0; cells with 1%-33% staining were scored as 1; cells with 34%-66% staining were scored as 2; cells with 67%-100% staining were scored as 3. Additionally, the staining intensities were evaluated into four grades: 3 (strong), 2 (moderate), 1 (weak) and 0 (none). The final score was defined as the score of percentage classifications multiplied by intensity grades.
4
Western Blot Analysis of Cellular Proteins
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RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) was used to lyse cells and liver tissue. Protein concentrations were quantified using a BCA kit (Beyotime Biotechnology, Shanghai, China). Proteins were separated by sodium dodecyl sulfate (SDS)-PAGE on 10% gels and transferred to poly-vinylidene difluoride (PVDF) membranes (Sigma-Alcdrih, St. Louis, MO, USA). After overnight incubation with various primary antibodies, including anti-HMGCL (1:1000, 16898-1-AP, Proteintech), GAPDH (1:2000, 10494-1-AP, Proteintech), DPP4 (1:1000, YT5707, Immunoway), H3 (1: 1000, 17168-1-AP, Proteintech), H3K9ac (1: 1000, ab32129, Abcam), H4 (1: 1000, 16047-1-AP, Proteintech), H4ac (1: 1000, 39026, Activemotif), Pan anti-acetyllysine (1: 1000, PTM-105, Jingjie PTM BioLab), Anti-LC3 (1: 1000, 4599, Cell Signaling Technology), Anti-P62 (1: 1000, 16177, Cell Signaling Technology), P16 (1: 1000, ab189034, Abcam), P21 (1: 1000, ab107099, Abcam), NOX1 (1: 1000, 17772-1-AP, Proteintech), EGFR (1: 1000, 66455-1-Ig, Proteintech). Then incubated for 2 h in the presence of secondary antibody (1: 2000, A0208, Beyotime) and washed 3 times with TBST for 5 min. The signals were detected using the ECL chemiluminescence system and analyzed by ImageJ Lab software.
5
Tissue Histology and Fibrosis Evaluation
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For tissue histology, the samples were immediately fixed in 4% PFA in PBS at 4°C for 16 h. The next day, samples were cryoprotected in a 30% sucrose solution in PBS at 4°C for 16 h, embedded in OCT compound, and stored at -80°C. Samples were then cut and mounted, and the different downstream assays were performed. Immunofluorescence staining was performed with antibodies that recognized phosphorylated EIF2A (S51) (Abcam, ab131505), CDKN2A/p16INK4a (Abcam, ab54210), or p21 (Abcam, ab107099). Collagen deposition was evaluated by staining the specified tissues with Masson's trichrome. For fibrosis evaluation, the relative area of fibrosis was calculated by measuring the area positive for Masson's trichrome (blue) divided by the area of total tissue (dark red). All the measurements were performed with ImageJ in a randomly selected field for each of the mice included in the analysis. All of the tissue staining procedures were performed commercially by iHisto (iHisto.io).
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