After the treatments, the cells were lysed on ice for 20 min with a mixture of 100 μl RIPA buffer (Fdbio science, Hangzhou, China), 1 μl protease inhibitor (Fdbio science) and 1 μl phosphatase inhibitor (Fdbio science) and centrifuged at 14,000 g for 25 min. Protein concentrations were determined using a BCA Total Protein Assay Kit (CWBIO, China). Protein samples (40 μg/lane) were separated on SDS-PAGE gels (8% or 12%, Epizyme, China) and electrotransferred to PVDF membranes. The blots were incubated with the following primary antibodies: anti-NLRP3 (1:500, A12694, ABclonal), anti-GSDMD (1:500, 39754, CST), anti-p20 (1:500, AF4005, Affinity), anti-CHOP (1:500, ab11419, Abcam), anti-GRP78 (1:500, ab21685, Abcam), anti-COL2A (1:500, ab34712, Abcam), anti-Aggrecan (1:500, ab36861, Abcam), anti-MMP3 (1:500, ab52915, Abcam), anti-MMP13 (1:500, ab39012, Abcam), anti-ADAMTS5 (1:1000, ab41037, Abcam), and anti-SREBP1 (1:1000, ab28481, Abcam). The membranes were washed and incubated with the HRP-conjugated secondary antibodies (1:8000, ABclonal, China) for 1 h. The bands were developed with chemiluminescence reagents and imaged by a myECL imager (Syngene G:BOX ChemiXT4, United Kingdom). The experiment was repeated in triplicate, and the blots were quantified by ImageJ software. An antibody against β-actin (AC026, 1:1000; ABclonal) served as an endogenous control.
Ac026
Manufactured by ABclonal
Sourced in China, United States
AC026 is a high-quality laboratory equipment designed for general use in scientific research and experiments. It provides precise and reliable performance to support various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
As014, by ABclonal (12 mentions)
Pvdf membrane, by Merck Group (9 mentions)
Ab6721, by Abcam (6 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Polyvinylidene fluoride membrane, by Merck Group (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Ab125066, by Abcam (4 mentions)
Bca kit, by Beyotime (4 mentions)
Ab32503, by Abcam (4 mentions)
Anti β actin, by ABclonal (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
β actin, by ABclonal (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
A19062, by ABclonal (3 mentions)
A5786, by ABclonal (3 mentions)
Bca assay kit, by Beyotime (3 mentions)
F1804, by Merck Group (3 mentions)
Ab184699, by Abcam (3 mentions)
Ac002, by ABclonal (3 mentions)
106 protocols using ac026
1
Comprehensive Protein Analysis of Cellular Responses
2
Lung Protein Extraction and GPX4 Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were executed 24 h after CLP. The right lobe of the lung was taken for testing (n = 6 per group). Protein was extracted using a mixture of RIPA lysis buffer. Primary antibodies against GPX4 (catalog number: ab125066; Abcam, Cambridge, UK), β-actin (AC026; ABclonal, Wuhan, China) and anti-rabbit secondary antibody (Ab6721; Abcam) were used.
3
Western Blot Analysis of Redox Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From colon tissues and IEC6 cells, total proteins were isolated using a lysis buffer. These proteins were subjected to heat at 98 °C for a short duration of 10 min. Following this, they were electrophoretically separated on a 10% SDS-PAGE and subsequently electrotransferred onto PVDF membranes. After the transfer, the membranes were blocked with a 5% non-fat milk solution for an hour at ambient temperature. Various primary antibodies were then introduced: GPX4 (1:500, A1933, abclonal, Wuhan, China), SLC7A11 (1:500, A13685, abclonal, Wuhan, China), TFR1 (1:500, A5865, abclonal, Wuhan, China), Nrf2 (1:500, A0674, abclonal, Wuhan, China), HO-1 (1:500, A19062, abclonal, Wuhan, China), and NQO-1 (1:500, A19586, abclonal, Wuhan, China), which were incubated with the membranes overnight in a 4 °C environment. This was followed by a one-hour exposure to a secondary antibody (1:7000, AS003, abclonal, Wuhan, China). For normalization, β-actin (1:5000, AC026, abclonal, Wuhan, China) served as an internal reference protein to evaluate relative protein expression levels. The membranes’ imaging was eventually carried out, with optical densities of the bands of interest being quantified using the AlphaEaseFC software4.0.0 suite from Alpha Innotech Corp, 3040 Oakmead Village Drive, Santa Clara, CA, USA.
4
Western Blot Analysis of TAP1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from 769P and 786O cells using Western and IP lysis buffer (Beyotime Biotechnology, Jiang Su, China), as previously mentioned (40 (link)). Samples were separated by electrophoresis on 10% SDS gel (epizyme, Shanghai, China) and then transferred to a methanol-activated polyvinylidene fluoride (PVDF) membrane (Millipore, Shanghai, China). Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and then incubated with primary antibodies, anti-TAP1 (1:1,000, 11114-1-AP, proteintech, China), and anti-beta-Actin primary antibody (1:1,000, AC026, Abclonal, China) at 4°C for 8 hours. After washed with TBST for three times, membranes were incubated with secondary antibody goat anti-rabbit IgG conjugated with HRP (1:3,000, SA00001-2, Proteintech, China) at room temperature for 60 min. After three washes with TBST for each 10 mins, the bands were visualized using ECL-plus™ western blotting chemiluminescence kits (BD Biosciences, NJ, USA). Image J was used to analyze strip grayscale, and Actin was used as a reference.
5
Western Blot Analysis of Osteoblast Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein of MC3T3 cells were harvested with RIPA lysis buffer and quantified with a BCA assay kit (Beyotime). Proteins were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). These membranes were blocked with 5% bull serum albumin in Tris buffered saline with Tween 20 and incubated with the following primary antibodies at 4 °C overnight: cyclin D1 (1:500, A2708, ABclonal, Wuhan, China), SP7 (1:1000, ab94744, Abcam, Shanghai, China), OCN (1:650, A5786, ABclonal), Runx2 (1:500, A2851, ABclonal), P27Kip1 (1:500, A2692, ABclonal), and β-actin (1:100000, AC026, ABclonal). Horseradish peroxidase-conjugated anti-rabbit IgG (H + L) secondary antibodies were added (1:3000, AS014, ABclonal) and incubated at 25 °C for 1 h. The signals were detected using an ECL chemiluminescence kit (7Sea biotech, Shanghai, China) by Tanon 5200 (Tanon, Shanghai, China).
6
Antibody Validation for Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies raised against the following proteins were used at the indicated concentrations for western blotting (WB): rabbit anti-FLAG antibody (Sigma, F7425; 1:5000 for WB); rabbit anti-GRSF1 antibody (Abcam, ab205531; 1:1000 for WB); rabbit anti-Fibrinogen alpha chain antibody (Abcam, ab92572; 1:1000 for WB); rabbit anti-Fibrinogen beta chain antibody (Abcam, ab189490; 1:1000 for WB); mouse anti-Fibrinogen gamma chain antibody (Abcam, ab119948; 1:1000 for WB); rabbit anti-β-actin antibody (ABclonal, AC026; 1:10000 for WB); mouse anti-GAPDH antibody (ABclonal, AC002; 1:10000 for WB).
7
Western Blot Analysis of Metabolic and Immune Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed using published protocols [2 (link),3 (link),33 (link)] with antibodies directed against HK2 (A01389, Boster), PKM2 (BM4601, Boster), LDHA (DF6280, Affinity), iNOS (ARG56509, arigo), TNF-α (GTX110520, GeneTex), Arg-1 (610708, BD), IL-10 (DF6894, Affinity), MCP1 (A7277, ABclonal), α-tubulin (Tub α, GTX112141, GeneTex), and β-actin (AC026, ABclonal).
8
Liver Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from liver samples as previously described. Protein concentration was measured with the Pierce BCA Protein Assay kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instruction. Western blot analysis of ACC1 (4190, Cell Signaling Technology, USA, diluted 1:1000), FASN (3189, Cell Signaling Technology, USA, diluted 1:1000), SCD1 (2438S, Cell Signaling Technology, USA, diluted 1:1000), PPARγ (WL01800, Wanleibio, China, diluted 1:500), CD36 (BS7861, Bioworld, China, diluted 1:1000), FTL (A18051, Abclonal, China, diluted 1:1000), FPN (DF13561, Affinity, China, diluted 1:1000), ZIP14 (ab123988, Abcam, USA, diluted 1:1000) and DMT1 (ab55733, Abcam, USA, diluted 1:1000) were carried out following the manufacturer’s instructions. Tubulin-α (BS1699, Bioworld, China, diluted 1:5000) or β-Actin (AC026, ABclonal, China, diluted 1:100,000) was used as internal control.
9
Production and Characterization of Polyclonal Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monoclonal anti–β-actin antibody (AC026, ABclonal Technology, Wuhan, China), polyclonal anti-H3 antibodies (ab1791, Abcam, Cambridge, UK), polyclonal anti-βCBP antibodies (14 (link)), and polyclonal rabbit antibodies against L. migratoria ATF2 (GenBank: OQ360058) and PKCα (GenBank: QNS30453) were prepared in our laboratory using recombinant protein from Escherichia coli to immunize rabbits. Briefly, fragments encoding portions of ATF2 (amino acids 190 to 360 and 490 to 626) or PKCα (amino acids 165 to 315) were amplified and fused with pET-28a (Promega) to produce the recombinant protein in E. coli rosette cells by 0.1 mM isopropyl β-d -thiogalactoside induction. The target protein was purified using a Ni2+-NTA affinity column (GE Healthcare) and served as an antigen to generate rabbit polyclonal antibodies with ABclonal Technology (Wuhan, China). The polyclonal antibodies against pS-ATF2 were generated by GenScript (Nanjing, China). Antibodies against p-Ser/Thr, pS-PKC substrate, and p-Thr were purchased from Abcam and Cell Signaling Technology.
10
Western Blot Analysis of ADAR2 in Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins from the liver tissues were collected using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was elevated using the BCA Protein Assay Kit. Total proteins were separated with 10% SDS-PAGE gels, transferred into PVDF membranes, and incubated with the primary antibodies at 4 ℃ overnight. The primary antibodies were as follows: ADAR2 (diluted 1:1000; A16748, ABclonal, Wuhan, China) and β-actin (diluted 1:100,000; AC026, ABclonal, Wuhan, China). After incubation with the secondary antibody, the proteins were visualized using a luminescent imaging system (BioRad, Hercules, CA, USA) and enhanced using the ECL Chemiluminescent Kit (Thermo Pierce, Waltham, MA, USA). The analysis of the protein bands was carried out using Image J software.
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!