Nucleoprotein extracts were treated with RNase (Solabio, China) and incubated with antibodies for hnRNPK (1:50) or IgG for 24 h on a rotating wheel. Thereafter, the Sepharose-conjugated protein-A/G beads were added and incubated at 4°C for another 24 h on a rotating wheel. After extensive rinsing with cold PBS, the beads were boiled, and the precipitated proteins were separated via SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature and subsequently incubated with primary and secondary antibodies, and protein bands were detected via enhanced chemiluminescence. The primary antibodies used were as follows: hnRNPK (Proteintech, #11426-1-AP, 1:1000), IgG (CST, #2729, 1:1000), hnRNPA1 (Proteintech, #11176-1-AP, 1:5000), hnRNPR (Proteintech, #15018-1-AP, 1:1000), hnRNPU (Proteintech, #16365-1-AP, 1:5000), Histone H3 (Abclonal, #A2348, 1:1000) and β-Actin (Proteintech, #20536-1-AP, 1:2000).
Hnrnpa1
Manufactured by Proteintech
Sourced in China
HnRNPA1 is an RNA-binding protein that plays a role in the regulation of mRNA processing and transport. It is involved in the formation of ribonucleoprotein particles and the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Hnrnpk, by Proteintech (1 mentions)
Hnrnpu, by Proteintech (1 mentions)
Histone h3, by ABclonal (1 mentions)
β actin, by Proteintech (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
Protease inhibitor, by Solarbio (1 mentions)
Immobilon ecl substrate, by Merck Group (1 mentions)
β actin, by Bioss Antibodies (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Sumo1, by ABclonal (1 mentions)
Ecl chemiluminescence system, by Clinx (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Nr4a3, by Abcam (1 mentions)
Hnrnp k, by Abcam (1 mentions)
Sf3b2, by Proteintech (1 mentions)
Lamin b1, by Santa Cruz Biotechnology (1 mentions)
Parp 1, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
5 protocols using hnrnpa1
1
hnRNPK Protein Immunoprecipitation and Analysis
2
Kaempferol Modulates Cancer Pathways
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Kaempferol was purchased from Weikeqi (Chengdu, China) and stocked in dimethyl sulfoxide (DMSO) at 200 mM. Fluorouracil (5-Fu, ≥99.0%) was dissolved in double-distilled water (80 mM). In the experiment for kaempferol treatment, the blank control group (0 μM) represented DMSO with a concentration of 0.05%, which was less than 0.1%; its toxicity to cells was thus negligible. DMSO and 5-Fu were purchased from Solarbio (Beijing, China). Antibodies against ABCB1, PKM1, PKM2, hnRNPA1, hnRNPA2 and PTB were purchased from ProteinTech (Wuhan, China). Antibodies for GAPDH, ABCC1 and ABCG2 were obtained from ImmunoWay Biotechnology (Plano, TX, USA).
3
Western Blot Analysis of HNRNPA1
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Cells were harvested and lysed by using the RIPA buffer containing protease inhibitors (Solarbio, China), and subsequently, the concentration of the protein samples were quantified by using the Enhanced BCA Protein Assay Kit (Beyotime, China). Protein samples were loaded and separated by 10% SDS-PAGE, and subsequently, they were transferred onto 0.45 μm PVDF membranes (Millipore, USA). The membranes were soaked in PBST buffer containing 5% skim milk at 37°C for 1 h, and then washed with PBST buffer thrice for 10 min. Subsequently, the membranes were incubated with the primary antibodies specific for HNRNPA1 (1:1,500, Proteintech, China) and β-actin (1:2,000, Bioss, China) at 4°C overnight, respectively. The membranes were washed three times with PBST buffer and were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Signals of the protein bands were detected by utilizing Immobilon ECL substrate (Millipore, Germany) and Azure Gel Imaging Systems C500 (Azure Biosystems, USA). Band intensity of western blot was measured by Image J 1.8.0 software (Bethesda, USA). The β-actin protein was selected as loading control.
4
Western Blot Analysis of PBMC Lysates
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Whole-cell lysate was subjected to western blot analysis following a previously described procedure [14] . For the preparation of total cell lysates, the PBMCs were lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1.0% deoxycholate, 1% Triton X-100, 1 mM EDTA and 0.1% SDS. The samples were centrifuged (12,000×rpm, 5 min) and the supernatants were further analyzed on a 12% SDS-PAGE gel and subsequently transferred to a PVDF membrane (Millipore, Massachusetts, USA). The following antibodies were used for western blotting: Tubulin (1:1000, AC015), SUMO1 (1:500, A2130) and UBE2Q2 (1:500, A9992) and were purchased from ABclonal Biotechnology. HNRNPA1 (1:500, A12446) and TRA2B (1:500, 23832–1-AP) were purchased from Proteintech group. All the antibodies were originated from rabbit. The immunoreactive proteins were detected by ECL chemiluminescence system (Clinx, Shanghai, China) with default settings and the Tubulin was set as the normalized control.
5
Western Blot Analysis of Hematopoietic Proteins
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Cells were lysed in a buffer containing 100 mM Tris (pH 7.6), 1% Triton X-100, 150 mM NaCl, 0.1 mg aprotinin, 35 mg/ml PMSF 10 mM Na3VO4, 100 mM NaF, 10 mM Na4P2O7, and 4 mM EDTA (approximately 5 × 106 cells). Samples were centrifuged at 4°C for 20 min to remove cell debris. Protein concentration was measured using the Bradford Assay (Bio-Rad). Laemmli buffer containing 100 mmol/L of dithiothreitol was added to the protein extracts and heated at 100°C for 5 min. Samples were run on a 10% SDS-PAGE. After the run, the proteins were transferred to nitrocellulose membranes (Millipore). Membranes were immunoblotted with NR4A3 (Abcam, ab41918), HnRNPK (Abcam, ab32969), SF3B2 (ProteinTech, 10919-1-AP), HnRNPA1 (ProteinTech, 11176-1-AP), PARP1 (Santa Cruz, sc-56197), Lamin B1 (Santa Cruz, sc-6127), and GAPDH (Santa Cruz, sc-32233) antibodies. K-562 protein samples were obtained from three independent experiments, all bands are shown in the Supplementary Material
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