The Sf9 cell monolayer was gently washed twice with phosphate-buffered saline (PBS) (pH = 7.4) and fixed with 80% cold acetone for 30 min at 4 °C. Two kinds of antibodies, anti-His tag monoclonal antibody (CWBIO, Beijing, China) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse immunoglobulin G (IgG) (CWBIO, Beijing, China), were used for an incubation with the cell monolayer for 1 h at 37 °C successively. Then, the cell monolayer was washed three times with PBS with 0.05% Tween-20 (PBST) and was watched under a florescent microscope.
Anti his tag monoclonal antibody
Manufactured by CWBIO
Sourced in China
The Anti-His tag monoclonal antibody is a laboratory tool used for the detection and purification of recombinant proteins containing a histidine (His) tag. It specifically binds to the His tag, allowing for the identification and isolation of the target protein from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by CWBIO (1 mentions)
Tetramethylbenzidine tmb, by LGC (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Mg132, by MedChemExpress (1 mentions)
Bca protein quantification kit, by CWBIO (1 mentions)
5 protocols using anti his tag monoclonal antibody
1
Immunofluorescence Assay for His-tagged Proteins
2
SDS-PAGE and Western Blot of His-Tagged Proteins
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The H5 cells were harvested 72 h pi and were washed three times with PBS, counted, and resuspended in PBS at a concentration of 2 × 106 cell equivalents per milliliter. The cells were frozen and thawed twice for lysis. The crude lysate was mixed with a sample buffer and boiled for 5 min. After a simple centrifugation, the proteins in the supernatant were separated on polyacrylamide gel. After that, the polyacrylamide gel was stained using Coomassie brilliant blue solution for 2 h and was then de-stained for several hours for observation. In addition, another same polyacrylamide gel was transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Berkeley, CA, USA). The membrane was blocked with 5% skim milk in PBS at 4 °C overnight and probed with an anti-His tag monoclonal antibody (CWBIO, Beijing, China). Then, the membrane was probed with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (CWBIO, Beijing, China). After being washed with PBST three times, the membrane was developed using tetramethylbenzidine (TMB) (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA), a horseradish peroxidase substrate, according to the manufacturer’s instructions.
3
Recombinant Protein Production of Amphioxus GH and Zebrafish PRL
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The recombinant protein of amphioxus GHl (rGHl) was prepared as described by Li et al. [26 (link)]. Zebrafish recombinant GH (rzGH) was purchased from ProSpec (East Brunswick, NJ, USA). The recombinant protein of zebrafish PRL (rzPRL) was produced by the methods of Li et al. [26 (link)]. Briefly, the cDNA encoding mature PRL (25 to 210 amino acids) was amplified by PCR, and the PCR products were digested with EcorI and XhoI and subcloned into the plasmid expression vector pET28a (Novagen, Germany) cut with the same restriction enzymes. The plasmid constructed was verified by sequencing and transformed into the cells of Escherichia coli BL21 (DE3). The recombinant protein was expressed, purified, and refolded as described by Li et al. [26 (link)]. The purified protein was analyzed on a 12% SDS-PAGE gel and immunostained using anti-His-tag monoclonal antibody (CWBIO, China) as the primary antibody. All the protein concentrations were determined with BCA protein assay kit (Beyotime, China).
4
Cell-free Protein Degradation Assay
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Cell-free protein degradation assays were conducted as previously described (Wang et al., 2009 (link)) with some modifications. Total petal proteins at stage 3 were extracted with degradation extraction buffer (25 mM Tris–MES, pH 7.5, 1 mM MgCl2, 4 mM PMSF, 5 mM DTT) and the cell debris was removed by centrifugation at 12 000 g at 4 °C. The supernatant was kept for the degradation assays. The purified PsCHS protein was divided into two equal parts and incubated with the total proteins at 25 °C. The specific 26S proteasome inhibitor MG132 (MedChemExpress) with a final concentration of 40 μM was added into one of the two parts and DMSO was added to the other as control. Samples were taken at different time intervals to measure PsCHS protein abundance by western blot analysis using an anti-His-tag monoclonal antibody from immunized mouse (1:5000, CWBIO, Beijing, China).
5
Purification of recombinant PyasOBP2 protein
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The culture was diluted 1:100 with liquid LB, and incubated at 37°C until the OD600 reached a value of 0.6–0.8. Protein expression was induced by adding isopropyl-β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM into the culture, and allowed to last for 10 h at 18°C. The bacterial cells (500 ml) were collected by centrifugation (8000 g for 10 min, 4°C), and the cell pellet was suspended with the lysis buffer (50 mg/ml Lysozyme and 20 mM Tris-HCl buffer at pH 7.4). The suspension was sonicated on ice, and centrifuged (12000 g for 30 min, 4°C) for a second time. Recombinant PyasOBP2 was examined by Sodium Dodecyl Sulfate—Polyacrylamide Gel electrophoresis (SDS-PAGE). Protein present in the supernatant was purified with a Ni-NTA His·Bind Resin column (7 Sea Biotech, Shanghai, China). The purified protein was assessed by SDS-PAGE, identified with the anti-His tag monoclonal antibody (Cwbio biotech, Beijing, China) by the Western Blot analysis, and desalted in a dialysis buffer (20 mM Tris-HCl at pH 7.4). To avoid confounding effects on subsequent experiments, His-tag was removed from the protein using a recombinant enterokinase (rEK) (Yeasen Biotech, Shanghai, China), and the concentration of the protein was assayed by the BCA protein quantification kit (Cwbio biotech, Beijing, China).
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