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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.

 

Concanavalin A
T4004-5mg	TargetMol Chemicals	5mg	Ask for price
Description: Concanavalin A

Concanavalin A
RM4157-100MG	EWC Diagnostics	1 unit	EUR 116.59
Description: Concanavalin A

Concanavalin A
RM4157-25MG	EWC Diagnostics	1 unit	EUR 33.38
Description: Concanavalin A

Concanavalin A
inh-cona-2	InvivoGen FR	2 x 100 mg	EUR 143.85

Concanavalin A
MBS3607807-25mg	MyBiosource	25mg	EUR 200

Concanavalin A
MBS3607807-5x25mg	MyBiosource	5x25mg	EUR 580

Concanavalin A
MBS5750377-100mg	MyBiosource	100mg	EUR 175

Concanavalin A
MBS5750377-25mg	MyBiosource	25mg	EUR 145

Concanavalin A
MBS5750377-50mg	MyBiosource	50mg	EUR 150

Concanavalin A
MBS5750377-5x100mg	MyBiosource	5x100mg	EUR 630

Concanavalin A IV
abx082153-25mg	Abbexa	25 mg	EUR 309.6

Concanavalin A IV
abx082153-100l	Abbexa	100 µl	EUR 162.5

Concanavalin A IV
abx082153-1ml	Abbexa	1 ml	Ask for price

Concanavalin A IV
abx082153-200l	Abbexa	200 µl	Ask for price

Concanavalin A (ConA)
05-0106-100	Medicago	100 mg	EUR 91.2

Concanavalin A (ConA)
05-0106-1000	Medicago	1 g	EUR 238.8

Concanavalin A (ConA)
05-0106-10000	Medicago	10 g	Ask for price

Concanavalin A (ConA)
05-0106-100000	Medicago	100 g	Ask for price

Concanavalin A (ConA)
05-0106-250	Medicago	250 mg	EUR 105.6

Concanavalin A (ConA)
E16FXP010	EnoGene	1 mg	EUR 86.67
Description: Available in various conjugation types.

Concanavalin-A Antibody
MBS7152270-INQUIRE	MyBiosource	INQUIRE	Ask for price

BioMagPlus Concanavalin A
BP531-10	Bangs Laboratories	10 ml	EUR 1399.73
Description: BioMagPlus Concanavalin A is magnetic particles with affinity binding proteins may be coated with the primary antibody or biotinylated molecule

BioMagPlus Concanavalin A
BP531-3	Bangs Laboratories	3 ml	EUR 621.95
Description: BioMagPlus Concanavalin A is magnetic particles with affinity binding proteins may be coated with the primary antibody or biotinylated molecule

Concanavalin A ELISA Kit
20-abx052181	Abbexa	Ask for price Ask for price Ask for price	10 × 96 tests 5 × 96 tests 96 tests

Concanavalin A ELISA Kit
ECP7827	Genovis AB	96 Tests	EUR 479

Concanavalin A (Con A) Antibody
20-abx101152	Abbexa	Ask for price Ask for price Ask for price Ask for price Ask for price	100 ug 10 ug 1 mg 200 ug 50 ug

Concanavalin A agarose gel
EA-IP-014	Elabscience Biotech	2mL	EUR 242
Description: Primary Antibodies

Concanavalin A (ConA) Antibody
abx101152-100l	Abbexa	100 µl	EUR 287.5

Concanavalin A (ConA) Antibody
abx101152-1ml	Abbexa	1 ml	EUR 850

Concanavalin A (ConA) Antibody
abx101152-200l	Abbexa	200 µl	EUR 375

Anti- Concanavalin A Antibody
GWB-5B3EDC	GenWay Biotech	1 ml	Ask for price

Concanavalin A Lectin (Con A) - Cy3
21761096-1	Glycomatrix	1 mg	EUR 148.5

Concanavalin A Lectin (Con A) - Cy5
21761160-1	Glycomatrix	1 mg	EUR 142.41

Concanavalin A Magnetic Beads
K1308-1	ApexBio	1ml	EUR 44.8
Description: Protein & Immunology|Protein Purification and isolation

Concanavalin A Magnetic Beads
K1308-5	ApexBio	5x1ml	EUR 196.8
Description: Protein & Immunology|Protein Purification and isolation

Concanavalin A Magnetic beads
EA-IP-014M	Elabscience Biotech	1mL	EUR 159
Description: Primary Antibodies

Concanavalin A Lectin (Con A) - Pure
22070010-1	Glycomatrix	250 mg	EUR 111

Concanavalin A Lectin (Con A) - Pure
22070010-3	Glycomatrix	25 mg	EUR 72.06

Concanavalin A Lectin (Con A) - Pure
22070010-4	Glycomatrix	50 mg	EUR 104.25

Concanavalin A, CF®350 Conjugate
29015	Biotium	5x1mg	EUR 189
Description: Lectin from Jack bean (Canavalia ensiformis)

Concanavalin A, CF®350 Conjugate
29015-1	Biotium	PK	EUR 189

Concanavalin A, CF®594 Conjugate
29017	Biotium	5x1mg	EUR 189
Description: Lectin from Jack bean (Canavalia ensiformis)

Concanavalin A, CF®594 Conjugate
29017-1	Biotium	PK	EUR 189

Concanavalin A, CF®633 Conjugate
29018	Biotium	5x1mg	EUR 189
Description: Lectin from Jack bean (Canavalia ensiformis)

Concanavalin A, CF®633 Conjugate
29018-1	Biotium	PK	EUR 189

Concanavalin A, CF®680 Conjugate
29020-1	Biotium	PK	EUR 224

Concanavalin A, CF®770 Conjugate
29058	Biotium	5x1mg	EUR 224
Description: Lectin from Jack bean (Canavalia ensiformis)