Cells were lysed by NP-40 with protease inhibitors and phosphatase inhibitors; the proteins were separated via SDS-PAGE electrophoresis, transferred onto nitrocellulose membranes, and then incubated with primary and secondary antibodies, respectively. Primary antibodies against JNK1/2/3 (YT2441) (1 : 1000) and phospho-JNK1/2/3 (phospho Thr183/Y185) (1 : 1000) were obtained from ImmunoWay Biotechnology (Newark, DE, USA). Primary antibodies against ERK (ab196883, 1 : 1000), p-ERK (ab50011, 1 : 5000), p38 (ab27986, 1 : 1000), and p-p38 (ab45381, 1 : 5000) were purchased from Abcam (Abcam, Cambridge, MA, United States). The JNK inhibitor SP600125 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The immunoreactive bands were detected with an enhanced chemiluminescence detection system from Pierce (Thermo Fisher Scientific Inc., Rockford, IL, USA). Protein band was analyzed with Quantity One software (Bio-Rad Laboratories, Life Science Research, CA, USA). Protein levels were normalized to that of the internal control β-actin purchased from Abcam (Cambrige, MA, USA) (ab8226, 1 : 1000).
Ab196883
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab196883 is a monoclonal antibody that specifically binds to the human CD11b protein. CD11b is a subunit of the integrin Mac-1, which is involved in various immune functions. The antibody can be used for the detection and study of CD11b in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab214362, by Abcam (2 mentions)
Ab18197, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab50011, by Abcam (1 mentions)
Ab45381, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab27986, by Abcam (1 mentions)
Sp600125, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ab31828, by Abcam (1 mentions)
Ab47363, by Abcam (1 mentions)
Ab13575, by Abcam (1 mentions)
Ab32023, by Abcam (1 mentions)
Ab205781, by Abcam (1 mentions)
Ab2324, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
5 protocols using ab196883
1
Western Blot Analysis of MAPK Pathway
2
Western Blot Analysis of Apoptosis Markers in Cardiomyocytes
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Proteins were extracted from the primary cardiomyocytes in RIPA buffer (1% Triton X-100, 150 mmol/L NaCl, 5 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH 7.0; Solarbio, China) supplemented with a protease inhibitor cocktail (Cat: I3786-1ML, Sigma). The cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to a PVDF membrane (Millipore Corporation, USA). After blocking with 8% milk in PBS, pH 7.5, the membranes were incubated with the following specific primary antibodies of Bax (ab32503), Bcl-2 (ab59348), cytochrome C (ab13575), Smac/Diablo (ab32023), cleaved-capase-3 (ab13847), cleaved-capase-9 (ab2324), p-p38 (ab47363), p38 (ab31828), p-ERK (ab214362), and ERK1/2 (ab196883; all at a dilution of 1:1000, Abcam, UK). After overnight incubation, the appropriate HRP-conjugated anti-rabbit IgG secondary antibody (ab205781, Abcam, all at a dilution of 1:5000) was subsequently applied and immunodetection was achieved using the ECL Plus detection system (Millipore Corporation) according to the manufacturer's instructions. Band intensity was quantified using Image Lab™ Software (Bio-Rad, China). GAPDH (ab8245, Abcam) was used as an internal control.
3
Protein Extraction and Western Blot Analysis
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The extraction of proteins from tissues or cells was implemented via radioimmunoprecipitation assay (RIPA, Sangon Biotech, Shanghai, China). Then, the protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (ThermoFisher, Waltham, USA). After the block by 5% non-fat milk, Western blot analysis was applied to determine the protein expression [14 (link)]. The membranes were incubated with the diluted primary antibodies (1:2000) and secondary antibodies (1:5000), respectively. Finally, the protein signals were achieved using an enhanced chemiluminescence (Amersham Pharmacia, Piscataway, USA). Primary antibodies against Ras (1:1,000, ab180772), phosphor-ERK1/2 (p-ERK1/2, 1:1,000, ab47339), ERK1/2 (t-ERK1/2, 1:3,000, ab196883), PCNA (1:2,000, ab18197), OCT4 (1:2,000, ab109183), hexokinase 2 (HK2) (1:2,000, ab227198), HMGA2 (1:2,000, ab97276), β-actin (1:2,000, ab8227), GAPDH (1:2,500, ab9485) and secondary antibodies (1:2,000, ab205718) were all obtained from Abcam. ImageJ software was used to evaluate the band density by densitometric analysis.
4
Immunohistochemical Analysis of Colon Tumor Xenografts
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Colon tumors from xenograft mice were fixed using formaldehyde (10%) at room temperature for 24 h and were then embedded in paraffin. The tissues were cut into 4-µm sections and mounted on glass slides. The sections were rinsed with PBS and placed in a solution containing primary antibodies directed against Ki67 (1:1,000; ab16667; Abcam), PCNA (1:1,000; ab18197; Abcam), Caspase3 (1:2,000; ab90437; Abcam), Caspase9 (1:1,000; ab32539; Abcam), Bcl-2 (1:1,000; ab692; Abcam), ERK (1:2,000; ab196883; Abcam), JNK (1:1,000; ab179461; Abcam), mTOR (1:1,000; ab87540; Abcam) and AKT (1:1,000; ab8978; Abcam) incubated overnight at 4°C. Subsequently, sections were incubated with the appropriate secondary antibodies, mouse primary IgG (1:1,500; ab6785; Abcam) and rabbit primary IgG (1:1,500; ab6721; Abcam), for 2 h at room temperature were added to specimens prior to visualization. The sections were then washed with PBS and observed by fluorescent video microscopy (BZ-9000; Keyence Corporation, Osaka, Japan) and a Ventana BenchMark automated staining system (Roche Applied Science, Penzberg, Germany) was used for observation of protein expression levels. A negative control was employed wherein mice were injected with 100 µl PBS.
5
Western Blot Analysis of Signaling Proteins
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In all, 30 μg proteins from cell homogenates were loaded in a 3–8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, blocked for 1.5 h using 5% nonfat dry milk in TBS-t and incubated with rabbit anti-NRF2 primary antibody (1:1000; Abcam, ab137550), rabbit anti-phospho-Akt (Ser473) (1:1000; Abcam ab81283), rabbit anti-Akt (1:1000; Abcam, ab126811), rabbit anti-phospho-ERK1/2 (Thr202 + Tyr204) (1:250; Abcam, ab214362), rabbit anti-ERK1/2 (1:500; Abcam, ab196883), rabbit anti-β-actin (1:2000; Abcam, ab8227) overnight at 4 °C. Then, the membrane was incubated for 1.5 h with a goat HRP-conjugated anti-rabbit secondary antibody (1:2000; Abcam, ab205718). Bands were detected by the Clarity™ Western ECL Blotting Substrate using a ChemiDoc MP system (Bio-Rad Laboratories Inc) and quantified by the Image Lab™ Software.
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