ADI has produced antibodies to a number of common fusion-tags to study expression of recombinant fusion proteins. In addition, we also provide ready to use affinity matrix (KLH and E.Coli proteins-Sepharose) for the removal of antibodies to KLH or E.coli proteins.


Antibodies and Conjugates for Myc-tag
 

Antibodies and Conjugates for Myc-tag
 

Antibodies and Conjugates for HA-tag 
 

Antibodies and Conjugates for GST-tag 

Antibodies and Conjugates for MBP-tag 
 

Antibodies and Conjugates for VSV-tag 
 

Antibodies and Conjugates for Thioredoxin 
 

Antibodies and Conjugates for Beta-Galactosidase 
 

Antibodies and Conjugates for GFP
 

Affinity Matrix for the removal of Fusion or Carrrier Proteins

Keyhole Limpet Hemocyanin, KLH-Agarose, Cat # KLH11-G; 1 ml; 5 ml (for the removal of KLH antibodies)

Antibodies to small peptide/haptens are generally raised by coupling to a large carrier proteins such as KLH. Antibodies are produced to both KLH and the peptide/hapten. Anti-KLH may give non-specific signals in various immunoassays. Anti-KLH antibodies can be removed by solid phase immunoaffinity column chromatography over the KLH-Agarose.

Source: KLH, isolated from the hemolymphs of the molllusk Megathura crenulata, belongs to a large family of giant respiratory proteins called hemocyanins, which are found in mollusks and arthropods. KLH was coupled to CNBR-activated Sepharose 4B at 3-4 mg protein/ml.

Form & Storage: The product is supplied as 1 ml settled gel in 1 ml of 0.01 PBS, 7.4 and 0.1% sodium azide

Do not freeze. Store at 4oC.

Binding capacity: Process 1-3 ml of high titer antiserum or 10 mg IgG over 1 ml of KLH-Agarose. The unbound fraction may be re-processed if necessary. The column can be used many times following standard binding, elution, and regeneration conditions.
We recommend processing approx. 1-5 ml antiserum per 1 ml of the beads or it can be scaled up accordingly. Load antiserum diluted 1:5 in PBS to adsorb anti-Ovalbumin at room temp. Collect unbound fraction containing Ovalbumin-depleted antiserum. It may be necessary to repeat this adsorption if the sample contain high concentrations of ant-Ovalbumin.

The column can be regenerated by passing 3 mls of 0.1M Glycine buffer, pH 2.5, and then immediately washing with PBS pH 7.4 with 10-20 volumes. Store column in PBS containing 0.05% azide at 4oC. DO NOT FREEZE the beads at any stage.

E.Coli Proteins-Agarose, Cat # EC11-G, (for the removal of E. coli protein's antibodies)

Recombinant proteins are very often expressed in E. coli and then purified for further studies or injected into animals to produce antibodies. One or more bacterial proteins very often contaminate the purified proteins. Antibodies are also produced to these minor contaminants. This may give non-specific signals in various immunoassays. Anti-E coli protein antibodies can be removed by solid phase immunoaffinity column chromatography over the E. coli Agarose.

Source: The E coli proteins were extracted from 3 E. coli strains (TG-1, Xl-1, and DH-57) coupled to CNBR-activated Sepharose 4B

Form & Storage: The product is supplied as 1 ml settled gel in 1 ml of 0.01 PBS, pH 7.4 and 0.1% sodium azide.

Do not freeze. Store at 4oC.

Binding capacity: Process 1 ml of high titer antiserum or 10 mg IgG over 1 ml of E. coli proteins-Agarose. The unbound fraction may be re-processed if necessary. The column can be used many times following standard binding, elution, and regeneration conditions.
We recommend processing approx. 1-5 ml antiserum per 1 ml of the beads or it can be scaled up accordingly. Load antiserum diluted 1:5 in PBS to adsorb anti-Ovalbumin at room temp. Collect unbound fraction containing Ovalbumin-depleted antiserum. It may be necessary to repeat this adsorption if the sample contain high concentrations of ant-Ovalbumin.

The column can be regenerated by passing 3 mls of 0.1M Glycine buffer, pH 2.5, and then immediately washing with PBS pH 7.4 with 10-20 volumes. Store column in PBS containing 0.05% azide at 4oC. DO NOT FREEZE the beads at any stage.

GST protein-Agarose, Cat # GST15R-AS; 0.5 ml (for the removal of GST antibodies or to purify GST binding proteins

Expression of genes in E. coli or yeast or baculovirus offers a convenient system to produce large amounts of recombinant proteins that may otherwise be difficult to isolate from natural cells and tissues. Very often antibodies to these newly identified proteins are not available to study its biochemical properties, monitor protein expression, and purification. In order to circumvent this problem, short pieces of well-defined peptides (Poly-His, Flag-epitope or c-myc epitope or HA-tag) or small proteins (bacterial GST, MBP, Thioredoxin, b-Galactosidase, VSV-Glycoprotein etc) are often cloned along with the target gene. Proteins are expressed as fusion proteins. Antibodies to these fusion-tags are already available to monitor fusion protein expression and purification. Therefore, fusion-tags serve as universal tags much like secondary antibodies. Many tags have their own characteristics. Poly-His-fusion proteins (6 x His) can bind to Nickel-Sepharose or Nickel-HRP. GST-fusion proteins can bind to glutathione-Sepharose. Therefore, a high degree of purification of fusion protein can be achieved in just one affinity purification step. Purity of fusion proteins can be followed by Tag-antibodies. Very often, fusion proteins are directly injected into animals to generate antibodies. Some fusion tags can be removed later by treatment with enzymes to generate tag-free recombinant proteins.

Source of Antigen and Antibodies: Bacterial GST (Schistosoma japonicum, ~27 kda) was expressed in E. coli and purified (>97%). Purified GST was coupled to agarose at ~5 mg/ml of beads (Cat # GST15R-AS). The affinity matrix is supplied in PBS pH 7.4 containing 0.05% azide. The column has a binding capacity of approx. 2-5 mg anti-GST per ml of beads.

Store at 4oC. DO NOT FREEZE.

GST protein concentration must be optimized for each application under defined experimental conditions.
Suggested uses: Many recombinant proteins are expressed and purified as GST-fusion protein and then injected into animals to raise antibodies. Antibodies are produced against the GST protein and the fusion protein. Anti-GST antibodies may interfere with some analyses. The GST-agarose column (Cat # GST15R-AS) can be used to remove the anti-GST antibodies.

We recommend processing approx. 1 ml antiserum per 0.25 ml of the beads or it can be scaled up accordingly. Load antiserum diluted 1:5 in PBS to adsorb anti-GST at room temp. Collect unbound fraction containing GST-depleted antiserum. It may be necessary to repeat this adsorption if the sample contain high concentrations of ant-GST.

The column can be regenerated by passing 3 mls of 0.1M Glycine buffer, pH 2.5, and then immediately washing with PBS pH 7.4 with 10-20 volumes. Store column in PBS containing 0.05% azide at 4oC. DO NOT FREEZE the beads at any stage.
 

Chicken Egg-Ovalbumin-Agarose, Cat # OVA15-AS; 1 ml (for the removal/or purification of ovalbumin antibodies)

Expression of genes in E. coli or yeast or baculovirus offers a convenient system to produce large amounts of recombinant proteins that may otherwise be difficult to isolate from natural cells and tissues. Very often antibodies to these newly identified proteins are not available to study its biochemical properties, monitor protein expression, and purification. In order to circumvent this problem, short pieces of well-defined peptides (Poly-His, Flag-epitope or c-myc epitope or HA-tag) or small proteins (bacterial GST, MBP, Thioredoxin, b-Galactosidase, VSV-Glycoprotein etc) are often cloned along with the target gene. Proteins are expressed as fusion proteins. Antibodies to these fusion-tags are already available to monitor fusion protein expression and purification. Therefore, fusion-tags serve as universal tags much like secondary antibodies. Many tags have their own characteristics. Poly-His-fusion proteins (6 x His) can bind to Nickel-Sepharose or Nickel-HRP. GST-fusion proteins can bind to glutathione-Sepharose. Therefore, a high degree of purification of fusion protein can be achieved in just one affinity purification step. Purity of fusion proteins can be followed by Tag-antibodies. Very often, fusion proteins are directly injected into animals to generate antibodies. Some fusion tags can be removed later by treatment with enzymes to generate tag-free recombinant proteins.

Source of Antigen and Antibodies: Purified chicken egg ovalbumin was coupled to agarose at ~5 mg/ml of beads (Cat # OVA15-AS) using CNBR-activated agarose beads. The affinity matrix is supplied in PBS pH 7.4 containing 0.05% azide. The column has a binding capacity of approx. 5-10 mg anti-Ovalbumin per ml of beads. Typically, this may corresponds to 5-10 ml antiserum containing antibodies to Ovalbumin. The anti-Ovalbumin IgG may vary and therefore, binding capacity of the Ovalbumin-agarose must be evaluated for each batch of antiserum,

Store at 4oC. DO NOT FREEZE.

Suggested uses: Many antibodies are made to small peptides or haptens that are injected as Ovalbumin-conjugates. Antibodies are produced against the carrier protein (Ovalbumin) and the coupled peptide or hapten. Anti-Ovalbumin antibodies may interfere with some analyses. The Ovalbumin-agarose column (Cat # OVA15-AS) can be used to remove the anti-Ovalbumin antibodies using standard antibody purification techniques.

We recommend processing approx. 1-5 ml antiserum per 1 ml of the beads or it can be scaled up accordingly. Load antiserum diluted 1:5 in PBS to adsorb anti-Ovalbumin at room temp. Collect unbound fraction containing Ovalbumin-depleted antiserum. It may be necessary to repeat this adsorption if the sample contain high concentrations of ant-Ovalbumin.

The column can be regenerated by passing 3 mls of 0.1M Glycine buffer, pH 2.5, and then immediately washing with PBS pH 7.4 with 10-20 volumes. Store column in PBS containing 0.05% azide at 4oC. DO NOT FREEZE the beads at any stage.

Bovine Serum Albumin (BSA)-Agarose, Cat # BSA15-AS; 1 ml (for the removal/or purification of ovalbumin antibodies)

Expression of genes in E. coli or yeast or baculovirus offers a convenient system to produce large amounts of recombinant proteins that may otherwise be difficult to isolate from natural cells and tissues. Very often antibodies to these newly identified proteins are not available to study its biochemical properties, monitor protein expression, and purification. In order to circumvent this problem, short pieces of well-defined peptides (Poly-His, Flag-epitope or c-myc epitope or HA-tag) or small proteins (bacterial GST, MBP, Thioredoxin, b-Galactosidase, VSV-Glycoprotein etc) are often cloned along with the target gene. Proteins are expressed as fusion proteins. Antibodies to these fusion-tags are already available to monitor fusion protein expression and purification. Therefore, fusion-tags serve as universal tags much like secondary antibodies. Many tags have their own characteristics. Poly-His-fusion proteins (6 x His) can bind to Nickel-Sepharose or Nickel-HRP. GST-fusion proteins can bind to glutathione-Sepharose. Therefore, a high degree of purification of fusion protein can be achieved in just one affinity purification step. Purity of fusion proteins can be followed by Tag-antibodies. Very often, fusion proteins are directly injected into animals to generate antibodies. Some fusion tags can be removed later by treatment with enzymes to generate tag-free recombinant proteins.

Source of Antigen and Antibodies: Purified BSA was coupled to agarose at ~10 mg/ml of beads (Cat # BSA15-AS) using CNBR-activated agarose beads. The affinity matrix is supplied in PBS pH 7.4 containing 0.05% azide. The column has a binding capacity of approx. 5-10 mg anti-BSA per ml of beads. Typically, this may corresponds to 5-10 ml antiserum containing antibodies to BSA. The anti-BSA IgG may vary and therefore, binding capacity of the BSA-agarose must be evaluated for each batch of antiserum,

Store at 4oC. DO NOT FREEZE.

Suggested uses: Many antibodies are made to small peptides or haptens that are injected as BSA-conjugates. Antibodies are produced against the carrier protein (BSA) and the coupled peptide or hapten. Anti-BSA antibodies may interfere with some analyses. The BSA-agarose column (Cat # BSA15-AS) can be used to remove the anti-BSA antibodies using standard antibody purification techniques.
We recommend processing approx. 1-5 ml antiserum per 1 ml of the beads or it can be scaled up accordingly. Load antiserum diluted 1:5 in PBS to adsorb anti-BSA at room temp. Collect unbound fraction containing BSA-depleted antiserum. It may be necessary to repeat this adsorption if the sample contain high concentrations of ant-BSA.

The column can be regenerated by passing 3 mls of 0.1M Glycine buffer, pH 2.5, and then immediately washing with PBS pH 7.4 with 10-20 volumes. Store column in PBS containing 0.05% azide at 4oC. DO NOT FREEZE the beads at any stage.

Immobilized albumin can also be used to affinity purify antibodies to BSA. It has also been used to remove from plasma a variety of substances (bilirubin, thyroxine, digitoxin etc) which binds to albumin. Elution of the bound substances can be achieved with 10-40 mg/ml of BSA or 50% ethanol or low (2.5) or high pH (11).
 

Recombinant Fusion Tags Information

Expression of genes in E. coli or yeast or baculovirus offers a convenient system to produce large amounts of recombinant proteins that may otherwise be difficult to isolate from natural cells and tissues. Very often antibodies to these newly identified proteins are not available to study its biochemical properties, monitor protein expression, and purification. In order to circumvent this problem, short pieces of well-defined peptides (Poly-His, c-myc epitope or HA-tag) or small proteins (bacterial GST, MBP, Thioredoxin, b-Galactosidase, VSV-Glycoprotein etc) are often cloned along with the target gene. Proteins are expressed as fusion proteins. Antibodies to these fusion-tags are already available to monitor fusion protein expression and purification. Therefore, fusion-tags serve as universal tags much like secondary antibodies. Many tags have their own characteristics. Poly-His-fusion proteins (6 x His) can bind to Nickel-Sepharose or Nickel-HRP. GST-fusion proteins can bind to glutathione-Sepharose. Therefore, a high degree of purification of fusion protein can be achieved in just one affinity purification step. Purity of fusion proteins can be followed by Tag-antibodies. Very often, fusion proteins are directly injected into animals to generate antibodies. Some fusion tags can be removed later by treatment with enzymes to generate tag-free recombinant proteins.

Fusion-tags are finding other ingenious application as well. Peptide can be made with 6X His and then noncovalently bound to Nickel-Sepharose. The Sepharose-bound peptide can be injected to generate antibodies. This method does not require covalent coupling of peptides to carrier proteins. There is a minimum modification of the peptide. In addition, there are no extraneous antibodies to carrier proteins. Conceivably, these tags-can also be utilized to monitor interaction of a peptide with other proteins, incubation with live cells or even develop competitive immunoassays.