For determining protein stability inside NPs, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were performed. Based on single bands for TIMP-1, it was assumed that the released protein was stable after release from NPs. For this, the supernatant collected at different time points was run in 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked overnight at 4°C in 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.02% Ps20 (TBST) containing 10% milk followed by washing three times in TBST for 10 minutes each. For detection, the membrane was incubated overnight in 1:200 dilution of mouse anti-TIMP-1 antibody (AF980; R&D Systems, Minneapolis, MN, USA), and rabbit polyclonal 6× Histag antibody (ab9108; Abcam, Cambridge, UK) in TBST at 4°C. The membrane was washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibody, and the bands were visualized using ECL Plus reagent (GE Healthcare Bio-Sciences, Uppsala, Sweden) substrate solution. Recombinant mouse TIMP-1 with a 6× Histag (WBC022; R&D Systems) was used as a standard for both Western blots. The blots shown in this study are representative replicates selected from at least three independent experiments.
Histag
Manufactured by R&D Systems
Sourced in United States
6× Histag is a protein tag used for the purification and detection of recombinant proteins. It consists of six consecutive histidine residues that can bind to nickel-charged affinity columns, allowing for the selective isolation of the tagged protein from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
His tag antibody, by Abcam (1 mentions)
Ecl plus reagent, by GE Healthcare (1 mentions)
Facscanto 2, by BD (1 mentions)
Ecl western blotting substrate, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hif 1α, by Proteintech (1 mentions)
Miseq platform, by Illumina (1 mentions)
4 protocols using histag
1
Evaluating Protein Stability in Nanoparticles
2
Quantifying SEMA7A-Plexin C1 Binding
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To evaluate the influence of glycosylation on the binding affinity of SEMA7A for Plexin C1, genetically modified HNSCC cells were seeded in 6-well plates and incubated with 2.5 μg/mL recombinant human Plexin C1 with a C-terminal 6-His tag (R&D SYSTEMS) for 24 h. The control group was treated with hIgG.Fc with a 6-His tag. The cells were then collected, washed and incubated with Alexa Fluor® 647‑conjugated Human IgG with a 6-His tag for 30 min. After final washing with cold PBS, the cells were subjected to analysis by flow cytometry (BD FACSCanto II).
3
Western Blot Analysis of Cellular Proteins
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Proteins were extracted from tissues or cells in RIPA buffer (NaCl 150 mM, Nonidet P-40 1%, sodium deoxycholate 0.5%, SDS 0.1%, Tris 25 mM, PH = 7.4) containing protease inhibitor cocktail (Merck). PVDF membrane was probed with primary antibodies at 4 °C overnight and incubated with HRP-conjugated secondary antibodies in room temperature for 1 hr. The protein bands were visualized by ECL Western Blotting Substrates (Bio-Rad). Antibodies used are: PDH2 (1:1000, Cell Signaling Technology, #4835 S, D31E11), iNOS (1:1000, Thermo Fisher Scientific, #PA3-030A), HIF-1α (1:1000, Proteintech, #20960-1-AP), HIF1α-OH-564 (1:1000, Cell Signaling Technology, #3434 S, D43B5), LDHA (1:1000, Cell Signaling Technology, #2012S), β-Actin (1:1000, Cell Signaling Technology, #4970 S, 13E5), His-tag (1:1000, R&D Systems, #MAB050R, AD1.1.10), rabbit IgG (1:2000, Cell Signaling Technology, #7074P2), mouse IgG light chain (1:2500, Cell Signaling Technology, #58802 S, D3V2A).
4
Antibody Discovery using Transgenic Rats
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Using genetically engineered transgenic rats (UniRat and OmniFlic) that express fully human IgG antibodies together with an NGS-based antibody discovery pipeline (TeneoSeek),25–28 (link) we have developed TNB-585. Methods for generating antibodies targeting CD3 in OmniFlic animals have been previously described.25 (link) For generating heavy chain only antibodies against PSMA, 12 UniRats were immunized with recombinant human PSMA protein fused to a his-tag (R&D Systems, Minneapolis, Minnesota, USA) for up to 8 weeks using either Titermax/Ribi or CFA/IFA adjuvant. Draining lymph nodes from all animals were then harvested, and total RNA was collected. cDNA samples containing the full heavy chain variable domain (VH) underwent next-generation sequencing using the MiSeq platform (Illumina, San Diego, California, USA) with 2×300 paired-end reads. Data from all animals were analyzed, and the most frequent 265 VH sequences were selected for cloning followed by expression in HEK 293 cells.
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