How does DSP crosslinker work?

How does DSP crosslinker work?

How does DSP work as a crosslinker? DSP is a homobifunctional, water soluble, cleavable, amine-reactive cross-linker. The amine-reactive ends are NHS-esters. This group reacts with primary amines, in proteins that translates to lysine groups and N-terminus amines.

What is DSS crosslinking?

DSS is a homobifunctional amine-specific protein crosslinking reagent based on NHS-ester reactive groups for selective conjugation of primary amines. DSS contains two NHS ester groups at both ends of a 8-carbon spacer that readily react towards primary amines at pH 7-9.

How do you dissolve DSS?

DSS is first dissolved in an organic solvent such as DMF or DMSO, then added to the aqueous crosslinking reaction. BS3, the water soluble analog of DSS is also available for applications that require a hydrophilic crosslinker (e.g. to effect cell-surface crosslinking).

How do you quench glutaraldehyde crosslinking?

Glutaraldehyde can be quenched through the addition of primary amine-containing buffers such as Tris. So before injecting your sample into the FPLC, add 5-10mM (final concentration) Tris at your desired pH and let it incubate for a few minutes.

Does glutaraldehyde denature proteins?

Synopsis. Glutaraldehyde reacts readily with various proteins in solution. This suggests that the proteins have not been denatured to any marked extent by the glutaraldehyde fixation. The rates of reactions increase with pH.

Is formaldehyde a solid liquid or gas?

Formaldehyde is a colorless, flammable gas at room temperature and has a strong odor. Exposure to formaldehyde may cause adverse health effects.

Why does DSS cause colitis?

Dextran sulfate sodium (DSS) is a sulfated polysaccharide with variable molecular weights. Administration of DSS causes human ulcerative colitis-like pathologies due to its toxicity to colonic epithelial cells, which results in compromised mucosal barrier function.

What is DSS inflammation?

DSS-induced Model For Studying Inflammatory Bowel Disease (IBD) Our DSS model is a chemically-induced study of Inflammatory Bowel Disease (IBD) using dextran sodium sulphate (DSS) in drinking water that induces ulcerative colitis-like inflammation.

What is the molecular weight of DSP crosslinker?

Molecular Weight: 404.42 Spacer Arm Length: 12.0 Å. General DSP Crosslinking Protocol. 1. Prepare a 50 mM solution of DSP crosslinker, by dissolving 10mg DSP in 495 μL of dry DMSO or dry DMF solvent.

How to use intracellular DSP crosslinking protocol?

Intracellular DSP Crosslinking Protocol. 1. Remove media by washing cells twice with non-amine containing buffer (i.e. 25 mM Sodium Phosphate, pH 7.4). 2. The crosslinking solution as noted in steps 1 and 2 above, then add the crosslinker solution to the cells in a final concentration of ~2 mM. 3.

How to reduce crosslinker and disulfide concentration in SDS-PAGE?

The first is by incubating the crosslinker in 10-50 mM DTT for 30 minutes at 37 degrees C. Secondly, the disulfIde can be reduced by incubation in 5% 2-Mercaptoethanol (2-ME) in SDS-PAGE sample buffer at 100 degrees C for 5 minutes.

What is the best buffer for ededc crosslinking reactions?

EDC crosslinking reactions must be performed in conditions devoid of extraneous carboxyls and amines. Acidic (pH 4.5 to 5.5) MES buffer (4-morpholino-ethane-sulfonic acid) is most effective, but phosphate buffers at pH ≤ 7.2 are also compatible with the reaction chemistry.