Agarose bound Phaseolus Vulgaris Leucoagglutinin (PHA-L)

AL-1113

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SKU Unit Size Price
AL-1113-2 2 ml
$174.00

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Description

Features:

  • Matrix is heat stable, cross-linked 4% agarose beads with a molecular exlusion of about 2x107 daltons
  • Bead diameter ranges in size from 45-165 microns
  • Matrix is stable in solutions at pH 3-11 as well as many organic solvents
  • Immobilized lectins are prepared using affinity purified lectins
  • Covalent attachment preserves lectin activity and minimizes conformational changes that might result in nonspecific or hydrophobic interactions
  • Hydrophilic spacer arm is inserted between the lectin and the matrix
  • Conjugated proteins are not leached off the beads by Tris or other routinely used buffers
  • No residual charges present after conjugation.  This minimizes non-specific binding to the matrix
  • Product supplied as a 1:1 suspension in buffer
  • 3 mg lectin/ml gel
  • Elution: 100 mM acetic acid or Glycoprotein Eluting Solution (ES-2100)

Specifications

More Information
Unit Size 2 ml
Applications Glycobiology, Affinity Chromatography
Matrix Conjugate Lectins
Sugar Specificity Galactose, Complex Structures
Conjugate Agarose

Documents

Product FAQs

I purchased an agarose bound lectin from Vector Labs. Do you have a protocol outline on how this may be applied in a column format?

Our agarose lectin products are supplied as hydrated matrix solutions in amber glass bottles. The agarose (bead) material will settle and you will see two phases in the tube supplied. The upper phase is buffer. A column can be prepared in a commercial plastic device such as Bio-Rad Cat # 732-6008 or an inverted Pasteur pipet with glass wool lightly packed in the neck to retain the agarose. 1) Draw (pipet) the desired amount of settled agarose-lectin (gel) from the stock bottle into the prepared column and let the buffer drain by gravity.(Sometimes an air bubble in the column tip prevents flow; tapping the column should get the flow started). 2) Wash the gel with 10 column volumes of buffer, such as HBS (10 mM HEPES, 0.15 M NaCl, pH 7.5) and discard the flow through. 3) Place a collection vessel (e.g. glass test tube) under the column tip and apply the glycoprotein-containing solution.Allow the solution to drain through using gravity. We recommend against pushing or pulling the material through the column. Retain the flow through material until the desired binding has been confirmed. 4) After sample application, wash column with 2-3 column volumes of buffer (or until the absorbance at 280nm is reduced to a satisfactory level) to remove unbound materials before elution. 5) Place a fresh collection vessel under the column tip.Apply the eluting solution again letting gravity do the work of moving the solution over the column. Note that in some cases, several column volumes of eluting solution may be required to achieved adequate release of bound material. 6) Following elution, the column can be prepared for reuse by washing with 10 column volumes of buffer. 7) If the column is to be stored, equilibrate the column with buffer containing 0.08% sodium azide. Cover the column with a plastic wrap, or similar, to prevent desiccation and keep at 4 degrees Celsius. The column will be stable for many months when stored under these conditions.

What are recommended conditions for using the agarose-lectin in chromatography?

The pH should be near neutral, the maximum pressure for packing the resin is 10 psi, and the maximum flow rate 3.5 ml/min.

Technical Information

Phaseolus vulgaris agglutinin is the name ascribed to a family of lectins, each of which consists of four subunits. There are two different types of subunits. One appears to be involved primarily in red cell agglutination and has been designated the E subunit (for erythroagglutinin). The other type is involved in lymphocyte agglutination and mitogenic activity and has been termed the L subunit (for leucoagglutinin). These subunits combine to produce five isolectins.

One of these isolectins has four E subunits and is designated PHA-E. PHA-E possesses strong hemagglutinating activity but is a poor mitogen. PHA-L, with four L type subunits, does not agglutinate red cells but is a potent mitogen. The other three isolectins, designated E3L1, E2L2, and E1L3, have erythroagglutinating and mitogenic activities proportional to the number of respective E or L subunits. We have termed the mixture of the five isolectins PHA (E+L).

PHA-L has been found to be an excellent, specific marker for use in anterograde neuronal tracing.

Agarose bound* PHA-L is prepared using our affinity-purified lectins. Heat stable, cross-linked 4% agarose beads with a molecular weight exclusion limit of about 2x107 daltons are used as the solid-phase matrix to which the lectins are covalently coupled. The attachment of the lectins to the beads is carefully controlled to preserve lectin activity and minimize conformational changes of the bound lectins that might result in nonspecific ionic or hydrophobic interactions. The technique we have developed to couple lectins to agarose beads inserts a hydrophilic spacer arm between the lectin and the matrix.

This coupling method provides several advantages over the traditional cyanogen bromide procedure:

  • Maximum carbohydrate binding activity of the coupled lectins is retained
  • Linkage is stable over a range of pH values
  • Conjugated proteins are not leached off the beads by Tris or other routinely used buffers
  • No residual charges are present after conjugation. This minimizes non-specific binding to the matrix.

Our agarose bound lectins are supplied at a constant concentration of lectin per ml of settled beads. The concentration for each lectin is selected to achieve the highest glycoconjugate binding capacity per mg of lectin present in the beads. Each lot is tested for its binding capacity using glycoproteins known to bind the lectin. This provides a guideline for the user and assures the quality of our agarose bound lectins.

Elution: 100 mM acetic acid

*3 mg lectin/ml gel