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How does biotin affinity purification work? Product FAQ

Answer

Immobilized avidin, streptavidin or Neutravidin is used to purify biotinlyated molecules with high stringency. The high affinity of these molecules for biotin requires non-reversible denaturation to release biotin itself (including boiling in SDS-PAGE sample buffer or 8M guanidine, pH 1.5).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Answer Id: E12905

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Article: Rapid Implementation of Novel Affinity Purification Product Literature

What are the benefits of CaptureSelect affinity products? Product FAQ

Answer

CaptureSelect affinity resins can make the purification of biomolecules that do not have a traditional affinity purification solution much more efficient. When a targeted, specific affinity purification solution does not exist for a biomolecule, the protein purification scheme can be very complex needing 4-5 chromatography steps. Many chromatographic steps need to be utilized to separate the product of interest from key process and product related impurities. As the number of required purification unit operations increases, the product yield decreases and is heavily impacted with each step added. Yield drives cost of goods, so not having an affinity purification solution can greatly impact the cost for biotherapeutic manufacturing. Using in-house capabilities and expertise, CaptureSelect Custom Services design product-specific affinity ligand based on single-chain, camelid-derived antibodies, and couple that ligand to a high-performance, cost-effective affinity resin. We currently offer off-the-shelf bioprocess resins for antibody fragments, biosimilars, and viral vectors. Custom ligands can be used for biomolecule purification, scavenging of challenging impurities, and quantitation or detection of biomolecules. Custom resins are suitable for use in a cGMP production process.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Answer Id: E12912

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After immobilizing my antibody, the first affinity purification of my protein worked well, but the protein failed to bind to the antibody during the second round of purification. What did I do wrong? Product FAQ

Answer

Low pH elution is the most commonly used method of elution for affinity purification, however, it could lead to denaturation of some antibodies which will negatively affect subsequent antigen binding. To prevent this from happening, after protein purification and column regeneration, immediately wash the column with neutralization or binding buffer and store at 4 degrees C in buffer containing an antimicrobial agent such as sodium azide.

Alternatively, use a near-neutral, high salt elution buffer such as Pierce Gentle Ag/Ab Elution Buffer (Cat. No. 21013, 21027, or 21030). For alternative elution buffers, please refer to this Tech Tip (https://tools.thermofisher.com/content/sfs/brochures/TR0027-Elution-conditions.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Answer Id: E12968

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L11059 AFFINITY ISOLATED ANTIBODIES SPECIFIC TO HUMAN IMMUNOGLOBINS ALKALINE Manual / Product Insert

  • Pub. No.: b4116ac277395ad26c3223c715def26cc8b689ed
  • Version: 12/08
Catalog #
  • H10308(Discontinued)
  • H16008(Discontinued)
  • H17008(Discontinued)
  • H15008(Discontinued)
  • H10008(Discontinued)
  • H14008(Discontinued)
  • H15708(Discontinued)

The protocol recommends using 10 - 75 µg of antibody with the Thermo Scientific Co-Immunoprecipitation Kit, is it possible to use a different amount? Product FAQ

Answer

Yes, the product manual includes “Table 1. Amounts of antibody, coupling resin, wash buffer and elution buffer to use” that allows researchers to scale their affinity purification using 5 - 1,000 µg of antibody. We do not recommend going below 5 µg of antibody. This was the smallest amount of antibody tested inhouse. The rationale to stop at 5 µg of antibody is that it was felt that an extremely strong affinity between the antibody and antigen would be required to get sensitive, reproducible results with less antibody to capture the antigen and interacting partners.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Answer Id: E13333

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In what applications can I use the Antibody Screening Spin Plates? Product FAQ

Answer

Protein A (Cat. No. 45202) and Protein G (Cat. No. 45204) Spin Plates for IgG Screening are for rapid, small scale, affinity-purification of polyclonal and monoclonal antibodies from serum, ascites and culture supernatants.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Answer Id: E8612

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Comparison of Affinity-Isolated and Non-Isolated Antibodies Used as capture Antibodies in ELISA Product Literature

What kind of animal serums or IgGs are adsorbed in preparation of ZyMAX grade antibodies? Product FAQ

Answer

When an antibody is listed as "ZyMAX Grade", the serum starting material is cross-absorbed after the antigen-affinity purification step against human serum proteins to reduce reactivity with human IgG. Note that we only sell 1 antibody that is designated as ZyMAX grade (Cat. No. 817140).

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E18732

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My sample lysates were made in RIPA buffer. Can I test these samples with your ELISA kits? Product FAQ

Answer

Yes, you can. The composition of the traditional 1X RIPA buffer is very similar to that of our Cell Extraction Buffer (Cat. No. FNN0011). Cat. No. FNN0011 is frequently used to prepare lysates for testing with our ELISA and Luminex kits. Our NP-40-based Cell Extraction Buffer (Cat. No. FNN0021) is also used. We recommend diluting lysates made with Cat. No. FNN0011 at least 10-fold in order to lower the SDS concentration to less than or equal to 0.01% (v/v) before adding the samples to the ELISA or Luminex assay.

Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.

Answer Id: E12625

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How do you typically detect expression of a recombinant fusion protein? Product FAQ

Answer

Western blot analysis is typically used to detect the expressed protein. We sell several antibodies against various epitopes, such as Xpress, HisG, V5, or C-terminal 6xHis. Additionally, His-tagged proteins can be purified using our ProBond Purification System via affinity purification.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E9695

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Are the CarboxylLink and DADPA UltraLink Resins available as kits? Product FAQ

Answer

CarboxyLink Coupling Resin is available as a kit (Cat. No. 44899), which contains sufficient components to prepare five reusable affinity columns. The resin is also available separately (Cat. No. 20266, 25 mL). DADPA UltraLink is available only as a kit (Cat. No. 53154). The kits do not contain buffers for affinity purification.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Answer Id: E8211

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Technical Bulletin: Comparison of Affinity-Isolated and Non-Isolated Antibodies Used as capture Antibodies in ELISA Product Literature

In what format are the antibody IgG purification products offered? Product FAQ

Answer

Protein A, Protein G, Protein A/G, and Protein L are all offered separately as purified protein, antibody purification kits, and covalently attached to agarose and other resins, magnetic beads and/or coated microplates. View the different product categories (https://www.thermofisher.com/us/en/home/life-science/antibodies/antibody-purification-kits-reagents/class-specific-affinity-purification-antibodies/igg-purification-kits.html) and product detail information. Additionally, please refer to Protein Immunoprecipitation (IP), Co- Immunoprecipitation (Co-IP), and Puldown Support for information pertaining to our Dynabeads magnetic bead-based kits (https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/protein-purification-isolation-support-center/protein-ip-coip-pulldown-support.html).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Answer Id: E12926

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I want to use the Antibody Clean-Up Kit (Cat. No. 44600) with a purified antibody that has a "protein based stabilizer". Do I need to first perform the Zeba desalting step A in the kit, or can I proceed directly to the BSA/Gelatin Removal step B? Product FAQ

Answer

The Zeba desalting step A is necessary as it results in a buffer exchange into the Melon Gel Purification Buffer. Having the antibody in this buffer is critical for the functionality of the Melon Gel used to remove protein.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Answer Id: E19617

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