BioHazards Bio-informe Fundamental Tools

G Protein Pathways, Part B: G Proteins and their Regulators

Cross-Linking Concanavalin A to SPR Chips
Concanavalin A is a tetrameric protein with four identical 28 kDa subunits. 29 The minimal structure exhibiting high affinity binding of carbohydrates is the dimeric form. The dimer form of ConA is an active stable structure, and the tetramer – dimer transition can be promoted by low pH or succinylation of the protein.30 For reasons of economy we use unmodified concanavalin A (Sigma). Because the dissociation of dimer from tetramer leads to a continual decline in the SPR baseline, our coupling procedures first promote the tetramer – dimer dissociation prior to linkage. To accomplish this, ConA is dissolved and stored at 4 ° in 100 mM sodium acetate, pH 4.8, prior to coupling. This also provides for optimal concentration of the ConA dimer by ionic attraction to the unactivated carboxyl groups. For limiting the ConA coupling the pH can be adjusted to 5.4 and still promote the dimer form.

However, extensive washing of the surface to allow complete tetramer dissociation is then necessary. Linkage of the ConA is accomplished by standard NHS / EDC amine coupling16 as described for streptavidin above, but with a modification in the running solution (Solution C, 50 mM MOPS, pH 7.5, 3 mM MgSO4, 150 mM NaCl with 10 μM CaCl2 and 10 μM MnCl2.

First, the surface is activated by injection of a mixture of 0.2 M EDC with 50 mM NHS in water at a flow rate of 5 μl / min, exposure time of 5–7 min. This is followed by injection of ConA at 0.1–0.3 mg / ml in 100 mM sodium acetate, pH 4.8, with an exposure time of 5–7 min. Activated carboxyls are then quenched by reaction with 1 M ethanolamine in water with an exposure time of 4 min. These procedures lead to the immobilization predominantly of dimeric ConA.

The immobilized ConA is quite stable, so that rhodopsin coupling does not have to proceed immediately. However, the coupling reactions are sufficiently reproducible and rapid that we find no advantage to prior preparation of ConA-modified surfaces. For experiments defining the independent binding of Gα or Gβγ described below, surfaces modified with 7000–12,000 RU of ConA have been utilized.

Under these conditions, the presence of immobilized tetrameric ConA is a significant factor. When high concentrations of ConA and / or aggressive NHS / EDC activation conditions are used, we routinely wash the ConA surface for 30–60 min with Solution C at a flow rate of 5 μl / min to allow for complete tetramer – dimer dissociation, prior to rhodopsin immobilization.

 

PRDM5 Polyclonal Antibody
A-2005	EpiGentek	EUR 754.26 EUR 117.70 EUR 284.90 EUR 470.80	100 µg 20 ul 50 ul 100 ul

PRDM10 Polyclonal Antibody
A-2010	EpiGentek	EUR 754.26 EUR 117.70 EUR 284.90 EUR 470.80	100 µg 20 ul 50 ul 100 ul

PRDM11 Polyclonal Antibody
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PRDM13 Polyclonal Antibody
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PRDM16 Polyclonal Antibody
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PRDM17 Polyclonal Antibody
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EZH1 Polyclonal Antibody
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HSF1 Polyclonal Antibody
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PRMT3 Polyclonal Antibody
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SET1 Polyclonal Antibody
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SET07 Polyclonal Antibody
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LSD1 Polyclonal Antibody
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HDAC10 Polyclonal Antibody
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PCAF Polyclonal Antibody
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Histone H3K9ac (Acetyl H3K9) Polyclonal Antibody
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Histone H3K14ac (Acetyl H3K14) Polyclonal Antibody
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Histone H3K18ac (Acetyl H3K18) Polyclonal Antibody
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Histone H3K23ac (Acetyl H3K23) Polyclonal Antibody
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Histone H3K56ac (Acetyl H3K56) Polyclonal Antibody
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Histone H3K4me1 (H3K4 Monomethyl) Polyclonal Antibody
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Histone H3K4me2 (H3K4 Dimethyl) Polyclonal Antibody
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Histone H3K4me3 (H3K4 Trimethyl) Polyclonal Antibody
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Histone H3K9me1 (H3K9 Monomethyl) Polyclonal Antibody
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Histone H3K9me2 (H3K9 Dimethyl) Polyclonal Antibody
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Histone H3K9me3 (H3K9 Trimethyl) Polyclonal Antibody
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Histone H3K27me1 (H3K27 Monomethyl) Polyclonal Antibody
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Histone H3K27me2 (H3K27 Dimethyl) Polyclonal Antibody
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Histone H3K27me3 (H3K27 Trimethyl) Polyclonal Antibody
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Histone H3K36me1 (H3K36 Monomethyl) Polyclonal Antibody
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Histone H3K36me2 (H3K36 Dimethyl) Polyclonal Antibody
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Histone H3K36me3 (H3K36 Trimethyl) Polyclonal Antibody
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Histone H3K79me1 (H3K79 Monomethyl) Polyclonal Antibody
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Histone H3K79me2 (H3K79 Dimethyl) Polyclonal Antibody
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Histone H3K79me3 (H3K79 Trimethyl) Polyclonal Antibody
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Histone H4K20me1 (H4K20 Monomethyl) Polyclonal Antibody
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Histone H4K20me2 (H4K20 Dimethyl) Polyclonal Antibody
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Histone H4K20me3 (H4K20 Trimethyl) Polyclonal Antibody
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Histone H3K27ac (Acetyl H3K27) Polyclonal Antibody
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CRISPR Cas9 Monoclonal Antibody [7A9]
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CRISPR/Cas9 (SaCas9) Monoclonal Antibody [6H4]
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CLIMP-63 Polyclonal Antibody
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