Anti-PEG IgG1 (CH2076, Silver Lake Research) and anti-PEG IgM (AGP4, IBMS) were used as test Ab and anti-Biotin IgG1 (Z021, Thermo Fisher) and anti-Vancomycin IgM (2F10, Santa Cruz) were used as control Ab. CH2076 was deglycosylated with rapid, non-reducing PNGase F (New England Biolabs) according to the manufacturer’s instructions. Deglycosylation was confirmed by SDS-PAGE gel with Coomassie stain and lectin-ELISA with biotinylated concanavalin A (B-1005, Vector Labs) and HRP-conjugated antibiotin IgG (033,720, Life Technologies). IgM was desialylated overnight with α2–3,6,8,9 Neuraminidase A (New England Biolabs) with 3 μL enzyme per 1 μg antibody at 37 °C. Desialylation was confirmed with lectin-ELISA with biotinylated elderberry bark lectin (B-1305, Vector Labs), biotinylated concanavalin A, and HRP-conjugated anti-biotin IgG. Due to the overnight incubation at 37 °C required to fully desialylate IgM and concerns it might denature IgM or otherwise reduce binding affinity to either PEG or the biogel matrix, we compared the trapping potency of desialylated IgM native IgM that was also incubated overnight at 37 °C.
Biotinylated concanavalin a
Manufactured by Vector Laboratories
Biotinylated concanavalin A is a lectin derived from the jack bean plant. It has a high affinity for glycosylated proteins and can be used to detect and isolate glycosylated biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated bsa, by Thermo Fisher Scientific (1 mentions)
Dasatinib, by Selleck Chemicals (1 mentions)
F actin, by Thermo Fisher Scientific (1 mentions)
Pyr 41, by Merck Group (1 mentions)
Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fluorescein wheat germ agglutinin, by Vector Laboratories (1 mentions)
Sequencing grade trypsin, by Promega (1 mentions)
β1 4 galactosidase, by New England Biolabs (1 mentions)
Fluorescein concanavalin a, by Vector Laboratories (1 mentions)
β1 3 galactosidase, by New England Biolabs (1 mentions)
O glycosidase, by Roche (1 mentions)
Maackia amurensis lectin 1, by Vector Laboratories (1 mentions)
Sambucus nigra lectin, by Vector Laboratories (1 mentions)
Anti biotin igg1, by Thermo Fisher Scientific (1 mentions)
B 1005, by Vector Laboratories (1 mentions)
Neuraminidase a, by New England Biolabs (1 mentions)
B 1305, by Vector Laboratories (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
5 protocols using biotinylated concanavalin a
1
Deglycosylation and Desialylation of Antibodies
2
Functionalization of Cantilevers for Biosensing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tipless cantilevers (Nanoworld
PNP-TR) were first O2 plasma treated for 20 min. The cantilevers
were then incubated in 0.5 mg/mL biotinylated BSA (Thermo Fisher Scientific)
for 12 h at 4 °C. The cantilevers were then rinsed with DPBS;
then, they were incubated in 0.5 mg/mL streptavidin (Sigma-Aldrich)
for 1 h at room temperature. The cantilevers were rinsed with DPBS,
then finally incubated in 0.1 mg/mL biotinylated concanavalin A (Vector
Laboratories) for 1 h at room temperature. The cantilevers were rinsed
and then stored in DPBS at 4 °C until use or up to 2 weeks.
PNP-TR) were first O2 plasma treated for 20 min. The cantilevers
were then incubated in 0.5 mg/mL biotinylated BSA (Thermo Fisher Scientific)
for 12 h at 4 °C. The cantilevers were then rinsed with DPBS;
then, they were incubated in 0.5 mg/mL streptavidin (Sigma-Aldrich)
for 1 h at room temperature. The cantilevers were rinsed with DPBS,
then finally incubated in 0.1 mg/mL biotinylated concanavalin A (Vector
Laboratories) for 1 h at room temperature. The cantilevers were rinsed
and then stored in DPBS at 4 °C until use or up to 2 weeks.
3
Dystroglycan Phosphorylation and Ubiquitination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dasatinib was obtained from Selleckchem (Munich, Germany) and Pyr-41 and MG132 from Sigma-Aldrich (Gillingham, UK). The following antibodies were used in WB and/or immunofluorescence (IF) applications. Non-phosphorylated β-dystroglycan [MANDAG2; WB 1:100, IF 1:100 (47 (link))], β-dystroglycan phosphorylated at tyrosine 892 [1709; WB 1:500 (13 (link),19 (link))], α-tubulin (WB 1:3500, Sigma-Aldrich), ubiquitin (WB 1:100, Enzo Life Sciences), biotinylated Concanavalin A (WB 1:4000, Vector Labs), western blots were detected by species-specific horseradish peroxidase conjugated to secondary antibodies or to streptavidin (1:5000, Sigma-Aldrich) and detected by ECL. In immunofluorescence, primary antibodies were detected by species-specific Alexa Fluor-conjugated secondary antibodies (IF 1:200, Molecular Probes) and F-actin was detected with rhodamine-conjugated phalloidin (IF 1:20, Molecular Probes).
4
Recombinant Human MR Glycosylation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human recombinant full-length MR with His-tag, expressed in mouse myeloma cell line NS0, was purchased from R&D systems, anti-human IgG Alexa-Fluor 488-conjugated from Jackson ImmunoResearch, and anti–penta-His Alexa-Fluor 488 conjugate from Qiagen. Biotinylated Concanavalin A, R. communis agglutinin-I, Maackia amurensis lectin-I, Sambucus nigra lectin, fluorescein Concanavalin A, fluorescein Wheat germ agglutinin, and Man-BSA were purchased from Vector Laboratories; neuraminidase and Protein A-agarose beads from Roche; O-glycosidase, PNGase F, α-galactosidase, β1,3-galactosidase, and β1,4-galactosidase from New England Biolabs; sequencing grade trypsin from Promega; Glucitol-polyacrylamide (PAA), Man3-PAA, and Glucitol-PAA biotin from Lectinity; GlcNAc and GlcNAc-BSA from Carbosynth. All other chemicals were purchased from Sigma-Aldrich if not indicated differently.
5
Immunoblotting Detection of Serum Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BSA conjugates
(24 , 25 , and 27–29 )
were subjected to sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and transferred
to a nitrocellulose membrane via semidry blotting. This membrane was
then incubated with blocking buffer [Tris-buffered saline containing
Tween 20; TTBS supplemented with 0.5% (w/v) BSA] for 1 h, prior to
incubation with either biotinylated concanavalin A (Vector), TEPC15
primary antibody (Sigma), human C-reactive protein (CRP; MPBio), or
human serum amyloid P (SAP; Merck) for 45 min. Thereafter, the membrane
was washed three times with TTBS before incubation with either alkaline
phosphatase-conjugated anti-biotin (Sigma), alkaline phosphatase-conjugated
anti-mouse IgA secondary antibody (Sigma), rabbit anti-CRP, or rabbit
anti-SAP for 45 min; in the case of the detection of pentraxins, alkaline
phosphatase- or peroxidase-conjugated anti-rabbit IgG was used. The
membrane was again washed three times with TTBS and then stained with
either SigmaFast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
or SigmaFast 3,3-diaminobenzidine in the dark. (All steps were performed
at room temperature; see theSupporting Information, page S42 , for dilutions.)
(
were subjected to sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and transferred
to a nitrocellulose membrane via semidry blotting. This membrane was
then incubated with blocking buffer [Tris-buffered saline containing
Tween 20; TTBS supplemented with 0.5% (w/v) BSA] for 1 h, prior to
incubation with either biotinylated concanavalin A (Vector), TEPC15
primary antibody (Sigma), human C-reactive protein (CRP; MPBio), or
human serum amyloid P (SAP; Merck) for 45 min. Thereafter, the membrane
was washed three times with TTBS before incubation with either alkaline
phosphatase-conjugated anti-biotin (Sigma), alkaline phosphatase-conjugated
anti-mouse IgA secondary antibody (Sigma), rabbit anti-CRP, or rabbit
anti-SAP for 45 min; in the case of the detection of pentraxins, alkaline
phosphatase- or peroxidase-conjugated anti-rabbit IgG was used. The
membrane was again washed three times with TTBS and then stained with
either SigmaFast 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
or SigmaFast 3,3-diaminobenzidine in the dark. (All steps were performed
at room temperature; see the
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!