Equal protein aliquots of BALF were incubated with PNA immobilized on agarose beads (Vector Laboratories, Burlingame, CA) to selectively enrich for PNA-binding proteins and the PNA-bound proteins resolved by SDS-PAGE as described above. The resolved proteins were transferred to PVDF membranes, and the membranes processed for MUC1-ED immunoblotting as above. Asialofetuin (1.0 µg) was used as a positive control for PNA-binding proteins whereas fetuin (1.0 µg) was used as a negative control.
Agarose beads
Manufactured by Vector Laboratories
Sourced in United States, France
Agarose beads are a matrix material used in various laboratory applications. They are made of agarose, a polysaccharide derived from red algae. Agarose beads provide a porous, hydrophilic structure that can be used for purposes such as chromatography, affinity purification, and other biochemical techniques.
Automatically generated - may contain errors
6 protocols using agarose beads
1
Enrichment and Detection of PNA-Binding Proteins
2
Capturing EGFR-containing Glycoproteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lycopersicon esculentum lectin (LEL) agarose beads (#AL-1113, Vector Labs, Burlingame, CA, USA) were used to analyze polylactosamine chains on glycoproteins. Cell lysates (0.5 mg) were incubated with 30 μL of LEL-conjugated beads to capture the lectin-glycoprotein complexes. Precipitated glycoproteins were eluted and subjected to Western blot analysis with an anti-EGFR antibody. The EGFR in total lysates was used as the internal loading control.
3
O-GlcNAc Enrichment and Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HCT116 cells and cyclin D1-FLAG-transfected HEK293 T cells were treated overnight with vehicle (DMSO) or ThG (1 μM) before lysis in RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate and 0.1% SDS). O-GlcNAc-modified proteins were enriched using the non-reducing terminal GlcNAc-specific lectin succinylated wheat germ agglutinin (sWGA) immobilized on agarose beads (Vector Laboratories, Clinisciences, Nanterre, France). Lysates (4 mg, 1 mg/ml) were incubated overnight at 4°C with 200 μl of sWGA-beads. Beads were centrifuged at 1,000 × g for 3 min, and then washed under vigorous stirring twice with 2 ml RIPA-0.1% SDS buffer and then three times with 2 ml RIPA-0.2% SDS buffer. Finally, beads were re-suspended in Laemmli buffer and stirred for 10 min using a vortex mixer before heating at 95°C for 7 min. Negative control was performed by adding 0.5 M free GlcNAc in the cell lysate before incubation with sWGA-beads. Eluted proteins were loaded on 10% SDS-PAGE to perform Western-blot analysis.
4
SNA Agarose Bead Pulldown Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sambucus Nigra Agglutinin bound Agarose beads (catalog AL-1303-2, Vector Laboratories) were washed twice with ice-cold PBS (catalog 10010049, Thermo Fisher Scientific). Pierce Protein A/G Plus Agarose beads (catalog 20423, Thermo Fisher Scientific) were used as control. After washing 50 μL of SNA Agarose beads or Protein A/G Plus Agarose beads, they were incubated with 500–1,000 μg cell lysates (collected as described above) in a total of 1 mL volume for 4 hours to overnight in a dark cold room (4°C) on a rotator. Beads were washed twice with ice-cold PBS followed by incubation with Pierce Lane Marker Reducing Sample Buffer (catalog 39000, Thermo Fisher Scientific) for 5 minutes at higher than 90°C for 5 minutes. The pulled lysates were subjected to IB as described above.
5
Identifying SA-binding Proteome in T. gondii
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain the SA-binding proteome of T. gondii, purified tachyzoites were suspended in phosphate-buffered saline (PBS) containing protease inhibitors and centrifuged at 12 000 rpm for 10 minutes. The supernatants of parasite lysates were incubated separately with SA-agarose (Vector laboratories) and agarose beads (Vector laboratories) in triplicate at 4°C overnight. The eluted proteins were subjected to liquid chromatography-sequential window acquisition of all theoretical fragment ion spectra (LC-SWATH) analysis. Proteins with an abundance ratio of SA-agarose/agarose ≥ 2 were selected as potential SA-binding proteins.
6
Immunoprecipitation from HEK293T and Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Forty hours post-transfection, HEK293T cells were washed in ice-cold PBS, harvested in Lysis buffer (50 mM Tris pH 7.5, 1% Triton X-100, 1.5 mM MgCl2, 5.0 mM EDTA, 100 mM NaCl2 and phosphatase/protease inhibitor cocktails (Santa Cruz and Sigma)) and tumbled for 1 hour to ensure complete lysis. The mouse brain was collected and immediately lysed in Lysis buffer. Lysates were then centrifuged at 10,000xg for 10 minutes and pellets were discarded. Bradford assays were performed to determine protein concentrations. Antibodies were added to lysates and gently tumbled overnight. Agarose beads (Vector Laboratories) were washed twice in Lysis buffer, added to the samples, and tumbled for 1 hour. Samples were spun down at 4000xg for 5 minutes and washed in Wash buffer (50 mM Tris, 0.1% Triton X-100, 1.5 mM MgCl2, 5.0 mM EDTA, 250 mM NaCl2 and phosphatase/protease inhibitor cocktails) three times. Lysates were kept on ice or at 4 °C throughout the entire experiment. Beads were boiled for 10 minutes in 2xLSB and samples were run on SDS-PAGE.
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!