Human sperm samples were capacitated by swim-up for 2 hours in Sperm Preparation Medium (Origio) and acrosome-reacted by calcium ionophore for 30 min at 37°C. Acrosome-reacted sperm were checked by PNA-FITC (1:300), for 15 min at RT after fixation by acetone:methanol for 5 min. Diluted MAIA/FcRL3 protein extracellular domain with His-tag (R&D Systems, 3126-FC) (1 μg/ml in PBS) was incubated with sperm for 1 hour at 37°C. Sperm suspension was fixed by acetone, and bound protein was visualized by His-tag antibody (Sigma-Aldrich) diluted 1:500 for 1 hour.
His tag antibody
Manufactured by Merck Group
Sourced in United States
His-tag antibody is a laboratory reagent used in protein purification and detection. It specifically binds to a short histidine-rich peptide tag that is commonly added to recombinant proteins to facilitate their isolation and identification.
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Lab products found in correlation
7 protocols using his tag antibody
1
Acrosome Reaction and MAIA/FcRL3 Binding
2
Verifying Recombinant Protein Expression
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At first, western blot was used to confirm the His-tagged recombinant protein. Briefly, the recombinant protein was size-separated on SDS-PAGE gel. Then, it was electroblotted on nitrocellulose membrane. The membrane was blocked overnight (4°C) with 3% skimmed milk in PBS. Subsequently, it was washed 3 times in a washing buffer (Tween 20 and PBS), incubated in His-tag antibody (Sigma, USA), and diluted in 1:1000 in PBS for 1 hour at room temperature. Afterwards, the membrane was washed 3 times, and DAB substrate (3, 3′-Diaminobenzidine) (Sigma, USA) was added to detect the recombinant protein.
3
Noncanonical Amino Acid Incorporation
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P-propargyloxy-l-phenylalanine (pPaF), p-azyl-phenylalanine (pAzF), p-acetyl-l-phenylalanine (pAcF), and p-benzoyl-l-phenylalanine (pBpF) were purchased from Sigma. His-tag antibody was purchased from Sigma. DNA plasmids used in CFPS were obtained from cultures of E. coli DH10B strain (Biomed Biotechnology, China) using Plasmid Mini kits (Omega Bio-Tek, America). All plasmids used in this experiment were sequence-verified. All linear PCR products were amplified with pfu high fidelity DNA polymerase (Beyotime Biotechnology, China). Primers were purchased from GENEWIZ Biotechnology with no modifications (Table S2 ).
4
Anti EpEX-scFv Expression Analysis
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The expression level of anti EpEX-scFv was analyzed by SDS-PAGE. After resuspending in 100 μL of 4× SDS sample buffer, the samples were heated for 5 min at 90 to 100 °C and 10 μL of each sample was loaded on 15.5% SDS-PAGE gel. After the electrophoresis, the gel was stained by Coomassie brilliant blue G-250. Quantification of the expressed protein was accomplished by image analysis of the SDS- PAGE gel using ImageJ software (NIH, MD). For western blot analysis, the proteins were electrotransferred from the gel into the polyvinylidene difluoride (PVDF) membrane using a wet Transblot (Bio-Rad, USA). The transferred membrane subsequently was blocked in 5% non-fat milk in tris-buffered saline-tween (TBST) for 1. The membrane was then washed three times by TBST and incubated overnight in His-tag antibody (Sigma, UK). Again, it was washed, then the membrane was incubated in the secondary antibody (anti-mouse horseradish peroxidase-conjugated immunoglobulin, Sigma, UK) for 2 h and then detected using a solution of 3,3’-diaminobenzidine (DAB; Sigma, UK).
5
Cellular Biotinylation of DKC1(1-514)
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The cellular biotinylation of the DKC1(1-514) protein followed the same approach as that described for CypD. DKC1(1-514) was expressed in BL21(DE3) in LB media and induced with 500 µM IPTG overnight at 18 °C. The PD-10 Desalting column was equilibrated with buffer U (50 mM HEPES pH 6.5, 750 mM KCl, 0.5 mM EDTA, and 0.5 mM TCEP).
All soluble cell lysates were concentrated and stored at −80 °C. The cellular biotinylated proteins were identified by Western blot, using a HisTag antibody (H1029 and A2429, Sigma-Aldrich), Streptavidin AP (21324, ThermoFisher Scientific, Waltham, MA, USA) or HRP Conjugate (SNN1004, ThermoFisher Scientific, Waltham, MA, USA).
All soluble cell lysates were concentrated and stored at −80 °C. The cellular biotinylated proteins were identified by Western blot, using a HisTag antibody (H1029 and A2429, Sigma-Aldrich), Streptavidin AP (21324, ThermoFisher Scientific, Waltham, MA, USA) or HRP Conjugate (SNN1004, ThermoFisher Scientific, Waltham, MA, USA).
6
GST Pull-down Assay for Protein Interactions
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GST Pull-down assay was performed as previously described36 (link). Pns10 or P8 gene of RDV was cloned into PGEX-3x to construct a plasmid expressing glutathione S-transferase (GST) fusion protein as bait. These two recombinant proteins were respectively expressed in the E. coli stain BL21, of which lysates were then incubated with glutathione-Sepharose beads (Amersham). Subsequently, recombinant protein of His-Tmod was added to the beads and incubated for 2 h, followed by washing the beads with PBS 10 times. Finally, elutes washed from the beads were analyzed with Western blotting assay using GST-tag and His-tag antibodies (Sigma), respectively.
7
Protein-protein interaction assay
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A GST pull-down assay was performed as previously described [57 (link)]. The Pns11 gene of RGDV was cloned in the pGEX-3x vector to construct a plasmid expressing the GST fusion protein as a bait (GST-Pns11). The full-length ORF of the VDAC from R. dorsalis was cloned into the pHM4 vector to construct a plasmid expressing the His fusion protein as a prey (His-VDAC). Recombinant proteins GST-Pns11 and GST were respectively expressed in the Escherichia coli stain BL21. Lysates were then incubated with glutathione-Sepharose beads (Amersham) and subsequently, with the recombinant protein His-VDAC. Finally, eluates were analyzed using GST-tag and His-tag antibodies (Sigma), respectively, in a Western blot assay.
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