Extraction of the cocoon shell protein was performed as previously described with slight modifications (Wang et al., 2013b (link)). Cocoons shell from WT and transgenic individuals were cut into small pieces and grind them into powder using the tissue grinder (Jingxin, China). 50 mg cocoon powder samples were immersed in 1 mL SDS-protein extraction solution overnight at 37°C. Protein samples were then subjected to SDS-PAGE (4%–15%), followed by staining with Fast Silver Stain Kit (Beyotime, China). For western blotting, the membranes were incubated with an anti-His antibody (1:3000, Sangon, China) and an anti-re-Dumpy specific peptide antibody (1: 1000, GenScript, China) as the primary antibodies. The specific polyclonal re-Dumpy antibody was raised in rabbits against the peptide “CRPAPPPEPTQSEYV” (GenScript, China). The signals were detected using the chemiluminescence imaging system (Clinx, China).
Anti his antibody
Manufactured by Sangon
Sourced in China
The Anti-His antibody is a laboratory reagent used for the detection and purification of recombinant proteins containing a histidine (His) tag. The antibody specifically binds to the His tag, allowing for the identification and isolation of the target protein from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fast silver stain kit, by Beyotime (1 mentions)
Chemiluminescence imaging system, by Clinx (1 mentions)
Tissue grinder, by Jingxin (1 mentions)
Gst resin, by Sangon (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Coomassie brilliant blue r 250, by Sangon (1 mentions)
4 protocols using anti his antibody
1
Cocoon Shell Protein Extraction
2
Recombinant Protein Expression and Interaction
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To produce glutathione S-transferase (GST)-KFB20 and GST-KFB39, the full-length coding sequences of KFB20 and KFB39 were separately amplified and cloned into the pGEX-4T-1 vector. To produce PAL1-His, the coding sequence of PAL1 was amplified and cloned into the pET-28a (+) vector. Protein expression was induced in the Escherichia coli BL21 (DE3) cell line by 0.3 mM IPTG and expressed GST fusion protein was purified using GST resin (C600031, Sangon Biotech, China) and expressed His fusion protein was purified using Ni-NTA agarose (30210, Qiagen, Germany).
To test the influence of CA on the interaction between PAL and KFB proteins, the GST pull-down assay was performed, and 140 μg GST-KFB20 or GST-KFB39 with 100 μg PAL1-His were added into the 1 mL reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM Na2EDTA, 10% glycerol, 0.1% Triton X-100, 1 × complete protease inhibitor cocktail, 1 mM DTT) containing different concentrations of CA, respectively. GST resin was used to bind GST-KFB20 or GST-KFB39. Proteins captured by GST resin were detected by immunoblotting with anti-His antibody (D410002, Sangon Biotech, China) and the Ponceau S staining method.
To test the influence of CA on the interaction between PAL and KFB proteins, the GST pull-down assay was performed, and 140 μg GST-KFB20 or GST-KFB39 with 100 μg PAL1-His were added into the 1 mL reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM Na2EDTA, 10% glycerol, 0.1% Triton X-100, 1 × complete protease inhibitor cocktail, 1 mM DTT) containing different concentrations of CA, respectively. GST resin was used to bind GST-KFB20 or GST-KFB39. Proteins captured by GST resin were detected by immunoblotting with anti-His antibody (D410002, Sangon Biotech, China) and the Ponceau S staining method.
3
Protein Expression and Western Blotting
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The samples collected during expression, purification, and cleavage were subjected to 12% SDS-PAGE, and then transferred to polyvinylidene fluoride (PVDF) membranes (Kong and Ryu, 2015 (link)). The PVDF membranes were blocked with 5% bovine serum albumin in phosphate buffered saline with Tween 20 (PBST) for 3 h and then incubated with anti-His antibody (1:3000) (Sangon Biotech, China) overnight, followed by peroxidase-affiniPure goat anti-mouse IgG (1:10000) (Sangon Biotech, China) for 1 h.
4
Silkworm Cocoon Protein Extraction
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Extraction of proteins from the cocoons of each transgenic silkworm strain was performed as previously described41 (link). Briefly, 1 undegummed cocoon was selected randomly and cut into pieces, grinding with liquid nitrogen until to powder. 20-mg cocoon samples were suspended in 400 μl of SDS-protein extraction solution for 3 h at room temperature. Subsequently, the protein-containing supernatant of each sample was collected by centrifugation at 15,000 rpm for 10 min at 4 °C and diluted with protein loading buffer for further assays. The cocoon protein samples were loaded at equal volume into 4–15% gradient SDS-PAGE gels (Sangon, Shanghai). Proteins were visualized by staining with Coomassie brilliant blue R-250 (Sangon). Proteins extracted from the cocoons were transferred onto a PVDF membrane (Immobilon-P, Millipore) after separation by SDS-PAGE. The membrane was blocked with 3% BSA in TBS-T (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20) and was then incubated with an anti-His antibody (1:5000 dilution, Sangon) as the primary antibody and peroxidase conjugate goat anti-rabbit IgG-HRP (1:5000 dilution, Sangon) as the secondary antibody. Signal detection was performed using a high-sensitivity ECL luminescence reagent kit (Sangon, Shanghai).
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