New method for analyzing phosphorylated proteins

Protein phosphorylation is an important post-translational modification and plays a key role in regulating gene expression and cell function. Phosphorylated antibodies are generally used to detect phosphorylation. In recent years, some new technologies and products have appeared on the market, which can realize the sensitive detection of phosphorylation.

Phos-tag â„¢ is one of them. It was originally developed by the Medical Molecular Functional Science Laboratory of Hiroshima University in Japan, and was later commercialized by Wako Corporation. Phos-tag â„¢ is a functional molecule that specifically binds to phosphate ions. Under neutral pH (physiological pH) conditions, it can be combined with anionic substituents, especially with divalent phosphate monoester anions.

The basic structure of Phos-tag â„¢

Phos-tag â„¢ technology takes advantage of this feature and replaces traditional enzyme immunoassay and radioisotope methods to capture substances with negative substituents, especially divalent phosphate monoester anions, improving the results of previous phosphorylation tests It is not a disadvantage of being particularly stable, but is a relatively stable technique for detecting phosphoric acid compounds so far.

At present, Phos-tag â„¢ products are mainly divided into four categories, which can be used for the separation of phosphorylated proteins (Phos-tag â„¢ Acrylamide), Western Blot detection (Phos-tag â„¢ Biotin), protein purification (Phos-tag â„¢ Agarose) and MALDI-TOF / MS (Phos-tag â„¢ Mass Analytical Kit).

Detection of phosphorylated protein

Phos-tag â„¢ Biotin is Phos-tag â„¢ combined with biotin, which can replace the phosphorylated antibody in western blot detection. Phos-tag â„¢ Biotin BTL-104 and BTL-105 can sensitively detect phosphorylated proteins on PVDF membranes. Now available in liquid form.

Features:

• radiation-free

• No need to close the PVDF membrane

• The specific binding of Phos-tag ™ has nothing to do with amino acid type and sequence

• Applicable to subsequent work such as immunoblotting and mass spectrometry

• Phos-tag ™ -BTL mother liquor can be stored stably for at least 6 months

• The experimental procedure is similar to the use of HRP labeled antibodies

Phosphorylated protein isolation

Phos-tag â„¢ Agarose provides an efficient method for separating and concentrating naturally phosphorylated proteins and phosphorylated peptides in biological samples (under physiological conditions).

Features:

• The buffer does not contain reducing agents and detergents

• Similar to affinity chromatography

• Purified phosphorylated protein within 1 hour

• Phos-tag ™ Agarose captures inorganic phosphate and large amounts of divalent phosphate bound to amino acids, sugars, and lipids such as Tyr, Thr, Ser, Asp, and His

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