Cells were lysed in RIPA buffer for 40 min at 4 °C. Removing insoluble material by centrifugation at 12,000 × g for 15 min at 4 °C, and the supernatants were used to assay protein levels. Protein samples (50 μg) were separated by electrophoresis on 12% and 5% SDS-PAGE gels using slab gel apparatus and then transferred to PVDF nitrocellulose membranes (Millipore, USA) blocked with 5% skim milk powder/Tween 20/TBST at room temperature for 2 h. Primary antibodies against Mark4 (ab124228) and GAPDH (ab181602) were purchased from Abcam (Cambridge, UK). Antibodies against IL-6 (ab6672), PGC1-α (ab54481), SOD (ab8866), MCP-1 (ab25124), IKKα (ab32041), phospho-IKKα (T23) (ab38515), NF-kB (ab32360), phospho-NF-kB (S337) (ab28849) and Cyt C (ab133504) were all form Abcam (Cambridge, UK). And NF-κB specific inhibitor PTDC was from Sigma (St. Louis, USA). Membranes were incubated with primary antibodies at 4 °C overnight and then incubated with the appropriate HRP-conjugated secondary antibodies (Boaoshen, China) for 2 h at room temperature. Proteins were visualized using chemiluminescent peroxidase substrate (Millipore, USA), and then the blots were quantified using ChemiDoc XRS system (Bio-Rad, USA) and Quantitative analysis of immune-blotted bands was performed using Quantity One software (Bio-Rad, USA).
Ab28849
Manufactured by Abcam
Sourced in United Kingdom
Ab28849 is a recombinant monoclonal antibody. It is produced in Escherichia coli.
Automatically generated - may contain errors
Lab products found in correlation
Ab220803, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Pvdf nitrocellulose membrane, by Merck Group (1 mentions)
Chemiluminescent peroxidase substrate, by Merck Group (1 mentions)
Ab38515, by Abcam (1 mentions)
Ab32041, by Abcam (1 mentions)
Ab6672, by Abcam (1 mentions)
Ab32360, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab25124, by Abcam (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Ab54481, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Ab2064, by Abcam (1 mentions)
Ab22555, by Abcam (1 mentions)
Ab13556, by Abcam (1 mentions)
Western blot special reagent, by Thermo Fisher Scientific (1 mentions)
Ab13847, by Abcam (1 mentions)
5 protocols using ab28849
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of TLR4 Signaling
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Total protein was extracted using RPPA lysis buffer (R0010, Solarbio) containing PMSF, and protein concentration was determined using a BCA kit (Thermo, USA). The sample was denatured by heating in the loading buffer at 100°C for 10 min. Then, 50 μg of protein was loaded, electrophoresed at 70 V for 3h, and transferred onto a PVDF membrane (ISEQ00010, Millipore, Billerica, MA, USA) at a constant flow of 150 mA. The membrane was blocked with 5% skim milk (Shanghai Xinyu Biotechnology Co., Ltd.) at room temperature for 4h at 20°C; washed with TBST; incubated with rabbit antibodies of TLR4 (ab13556, 1:500, Abcam, UK), MyD88 (ab2064, 1:500, Abcam, UK), NF-κB p50 (ab220803, 1 µg/mL, Abcam, UK), p-NF-κB p50 (phospho S337) (ab28849, 1 μg/mL, Abcam, UK), and GAPDH (ab22555, 1:2,000, Abcam, UK) overnight at 4°C; and washed with TBST again. Subsequently, the membrane was incubated with HRP-labeled goat anti-rabbit IgG antibody (Beijing Zhongshan Biotechnology Co., Ltd., diluted 1:5,000) for 2h, washed with TBST, and developed using the ECL fluorescence detection kit (Cat. No. BB-3501, Amersham, UK) on a Bio-Rad image analysis system (BIO-RAD, USA).
3
Western Blot Analysis of Ischemic Cortex Proteins
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The cells and ischemic cortex were collected, and the RIPA Lysis Buffer protein lysis solution (Roche, Basel, Switzerland) was added to isolate the total protein. Subsequently, 50 μg of total protein was loaded on 12% polyacrylamide gel for electrophoresis at 100 V for 2 hours, and then transferred to polyvinylidene fluoride membranes. After being blocked with 5% skimmed milk for 1 hour at room temperature, the membranes were washed three times with TBST (TBS containing 0.1% Tween20) for 10 min each time, and incubated with anti-STAT1 (1: 1000, ab92506, Abcam, Cambridge, MA, USA), anti-p-STAT1 (1: 1000, ab109461, Abcam), anti-NF-κB (1:1000, ab220803), Anti-p-NF-κB (1: 1000, ab28849), anti-Bcl-2 (1: 1000, ab59348), anti-Bax (1: 1000, ab32503), and anti-Caspase3 (1: 1000, ab13847) overnight at 4℃. After being rinsed with TBST, the membranes were incubated with horseradish peroxidase-labeled anti-rabbit secondary antibody (concentration 1: 3000) for 1 hour at room temperature. Afterward, the membranes were washed three times with TBST (10 min each time). Finally, Western blot special reagents (Invitrogen) were applied for color imaging, and ImageJ 1.44 software was used for density analysis.
4
Western Blot Analysis of TLR4 Pathway
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The lung tissues of four mice in each group were used to prepare tissue homogenate. Then total protein in the tissue was extracted by using RIPA lysis buffer containing PMSF (R0010, Solarbio). Protein concentration was measured according to the instructions of BCA kit (Thermo, U.S.A.) and adjusted. The protein was mixed with 5× loading buffer and denatured on the boiling water bath. SDS/PAGE was used to separate the denatured protein which was then transferred to PVDF membrane. Then the membrane was sealed with 5% skim milk at room temperature for 1.5 h and incubated at 4°C overnight with primary antibodies including rabbit anti-human TLR4 (1:1000, ab13867, Abcam, U.S.A.), MyD88 (1:1000, ab135693, Abcam, U.S.A.), NF-κB p50 (1 μg/ml, ab220803, Abcam, U.K.), p-NF-κB p50 (1 μg/ml, phospho S337, ab28849, Abcam, U.K.), Bax (1:1000, ab8805, Abcam, U.S.A.), Bcl-2 (1:1000, ab32124, Abcam, U.S.A.) and GAPDH (1:5000, ab9385, Abcam, U.S.A.). After the membrane was washed three times, it was added with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:10000, ab97051, Abcam, U.K.) and incubated for 2 h. Then the membrane was washed three times and developed. Imaging was implemented by Bio-Rad gel imager (Bio-Rad, U.S.A.). Relative protein expression = gray value of target protein band/gray value of GAPDH band.
5
Protein Extraction and Western Blot Analysis
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RIPA reagent (P0013B, Beyotime) was used for extracting proteins from HK-2 cells. Protein concentration was determined using a BCA Protein Assay kit (P0011, Beyotime). Protein samples were separated with 12% SDS-PAGE gel, transferred onto PVDF membranes, and blocked in 5% skim milk for 2 h at 37°C. Next, samples were treated with primary antibodies including anti-cleaved caspase-3 (1/500, ab32042, Abcam), anti-p50 (1/2000, ab283688, Abcam), anti-p-p50 (1/200, ab28849, Abcam), and anti-GAPDH (1/5000, ab8245, Abcam) overnight at 4°C and the secondary antibody (1/1000, ab96899, Abcam) at 37°C for 2 h. GAPDH is an internal control.
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