The extracellular portion of human PB1 was subjected to PCR with the following primers: Forward: 5′-CCCAAGCTTATGCCTGCTCTGGGCCCAGCTCTT-3′ and reverse: 5′-CCGGAATTCCCAAGCCCACCTGGGCTGCCACA-3′. The resulting product was cloned into the plasmid pSecTag2B (Invitrogen, Carlsbad, CA). This construct was transfected into 293 T cells growing in serum free media. Media was collected 65 hours post-transfection and purified with TALON metal affinity resin (Clontech Laboratories, Palo Alto, CA) according to manufacturer's instructions. Concentration and purity of the TALON eluates was determined by SDS PAGE analysis followed by silver staining (Amersham Life Science, Piscataway, NJ) and the Bio-Rad protein assay (Bio-Rad, Hercules, CA). In all cases, media collected from cells transfected with the empty pSecTag2B vector were used as control.
Silver staining
Manufactured by Cytiva
Sourced in Sweden
Silver staining is a laboratory technique used to detect and visualize proteins in gel electrophoresis. It involves the use of silver ions to selectively bind to proteins, resulting in a dark staining that can be observed under visible light. This method provides a sensitive and versatile approach for protein analysis and detection in various biological and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Talon metal affinity resin, by Takara Bio (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Psectag2b, by Thermo Fisher Scientific (2 mentions)
Dynabeads m 280, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Ultra low range molecular weight marker, by Merck Group (1 mentions)
Mmp 2, by Merck Group (1 mentions)
5 protocols using silver staining
1
Extracellular PB1 Protein Expression and Purification
2
Purification and Characterization of Soluble Sema4D
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Soluble Sema4D (sSema4D) was produced and purified as described previously [3 (link)]. Briefly, the extracellular portion of Sema4D was subjected to PCR and the resulting product cloned into the plasmid pSecTag2B (Invitrogen, Carlsbad, CA). This construct was transfected into 293T cells growing in serum free media. Media containing sSema4D was collected 65 h post-transfection and purified with TALON metal affinity resin (Clontech Laboratories, Palo Alto, CA) according to manufacturer’s instructions. Concentration and purity of the TALON eluates was determined by SDS PAGE analysis followed by silver staining (Amersham Life Science, Piscataway, NJ), comparing band intensity against a BSA standard curve on the same gel, and the Bio-Rad protein assay (Bio-Rad, Hercules, CA). In all cases, media collected from cells transfected with the empty pSecTag2B vector were used as control.
3
Tylophorine-Mediated Protein-RNA Interactome Elucidation
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HONE-1 cell lysates were incubated with biotinylated tylophorine at 4°C for 4 h. The biotinylated tylophorine-associated complexes were then pulled down using streptavidin Dynabeads M280 (Invitrogen) at 4°C for 1.5 h. The biotinylated tylophorine-bound proteins were eluted with Laemmli buffer and analyzed by electrophoresis on a 4%–20% gradient SDS-PAGE gel (W/H: 16 cm × 15 cm) and visualized by silver staining (Amersham Biosciences). The protein bands of interest were excised, subjected to in-gel digestion (see Supplemental Materials and Methods ), and analyzed by LC/MS/MS using a Thermo Scientific LTQ XL mass spectrometer (Thermo Scientific). In addition, western blot was used to identify the protein components from the pull-down. The biotinylated tylophorine-bound RNAs were eluted with TRizol Reagent (Invitrogen) and RT-PCR was further used for analyzing the associated components.
4
Determining Bacteriocin Molecular Weight
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The molecular weight of bacteriocin was determined by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, USA) at 100 V for 5 h on a 20% polyacrylamide gel with an ultra-low-range molecular weight marker (1,060–26,600 Da; Sigma-Aldrich), followed by silver staining (Amersham Biosciences, Sweden). Direct detection was then performed to determine whether the protein bands corresponded to bacteriocin [23 (link)].
5
MMP2-Mediated Tau Proteolysis Dynamics
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Full-length MMP2 (Sigma) was diluted to a final concentration of 100 g/ml and activated through its incubation with 1 mM APMA (4-aminophenylmercuric acetate) (Sigma) for 1 h at 37 • C. Afterwards, 2 g of the longest form of recombinant tau, which includes exons 2 and 3, and four microtubules-binding repeats (T40) (r-peptide) were incubated with 50 ng, 200 ng, and 500 ng of activated MMP2 in a buffer containing 50 mM Tris-HCl pH 7.5, 10 mM CaCl 2 , and 150 mM NaCl. The reaction was left at 37 • C for 3 h and then stopped by the addition of Laemmli SDS sample buffer and followed by boiling. The samples were subjected to 12% SDS-PAGE and the tau proteolytic profile was visualized first on silver staining (Amersham) and then by immunoblotting with both N-terminal tau (Millipore) and Tau 46 C-terminal (Abcam) antibodies diluted 1:1,000.
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