Dr. Astrid Sitte

Recent Posts

Co-immunoprecipitation troubleshooting

Posted by Dr. Astrid Sitte on Aug 27, 2021 5:21:16 PM

Co-immunoprecipitation (Co-IP) describes the isolation of a protein and its binding partners from a cell extract using a Nanobody or antibody that is bound to beads. The protein, that directly interacts with the Nanobody, or antibody beads is called “bait”. The binding partner that is indirectly precipitated is called “prey”.

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Topics: GFP-Trap, Immunoprecipitation, Co-IP

How to conduct a Co-immunoprecipitation (Co-IP)?

Posted by Dr. Astrid Sitte on Jul 30, 2021 2:39:52 PM

Co-IP describes the immunoprecipitation of a protein of interest and its interacting partners.

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Topics: GFP-Trap, Immunoprecipitation, Co-IP

Tips and tricks for immunoprecipitation of low abundant proteins

Posted by Dr. Astrid Sitte on Apr 14, 2021 4:03:04 PM

Protein abundance is crucial for immunoprecipitation

During an immunoprecipitation (IP), the protein of interest (POI) is pulled down with a specific antibody or Nanobody conjugated to beads such as agarose or magnetic agarose. Unbound sample content, for example, other proteins, cell debris, and lipids, is removed by washing, while the precipitated protein is enriched on the beads.

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Topics: GFP-Trap, GFP Immunoprecipitation

Overview of the most recent Spot Peptide-Tag & Spot-Nanobody publications

Posted by Dr. Astrid Sitte on Mar 25, 2021 12:46:40 PM

More and more publications are coming up using ChromoTek’s Spot-Tag system, the first peptide-tag specific Nanobody system for universal capture & detection applications.

Interaction of Spot-Nanobody (green) with Spot Peptide

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Conjugation of fluorescent dyes to Nanobodies

Posted by Dr. Astrid Sitte on Jan 27, 2021 2:01:35 PM

The conjugation of the binding domains of camelid heavy chain antibodies, also known as VHHs or Nanobodies, to fluorescent dyes is generating versatile tools for fluorescence microscopy applications like immunofluorescence (IF) and super resolution microscopy (SRM): The small size Nanobodies provide better penetration into tissue and dense cell compartments, plus the small label to epitope displacement provides higher resolution. In addition, the Nanobodies’ high affinity binding to epitope makes them attractive probes for other assays like ELISA.

ChromoTek, part of Proteintech group, provides multiple fluorescent dye (Alexa Fluor® and ATTO) conjugated Nanobodies that are optimized by application:

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Which beads should I use for my immunoprecipitation?

Posted by Dr. Astrid Sitte on Sep 16, 2020 9:42:57 AM

ChromoTek's Nano-Traps are optimized for the immunoprecipitation (IP) of proteins and their interacting factors. Nano-Traps comprise of a Nanobody/ VHH conjugated to beads. For 3 Nano-Traps, e.g. GFP-Trap®RFP-Trap®, and Spot-Trap®, we offer different types of beads:

  • Agarose beads
  • Magnetic Agarose beads
  • Magnetic Particles M-270

Immunoprecipitation with GFP-Trap.
I: Input, FT: Flow-Through, B: Bound

Based on the properties of the different beads (see table), we recommend:

  • Agarose beads for very low background and high binding capacity IP
  • Magnetic Agarose beads for magnetic separation and high binding capacity IP
  • Magnetic Particles M-270 for IP of very large proteins/complexes
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Topics: GFP-Trap, Immunoprecipitation, RFP-Trap, GFP Immunoprecipitation, GFP Nanobody, Spot-Trap

Characterization of COVID-19 antibodies by Bio-Layer Interferometry using Nano-CaptureLigands

Posted by Dr. Astrid Sitte on Jul 8, 2020 9:10:21 AM

Characterization of antibodies

Essential aspects of how antibodies (Abs) bind to their targets are their affinity and binding kinetics. In order to determine these parameters, different biophysical methods such as Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI), MicroScale Thermophoresis (MST), or Enzyme-linked Immunosorbent Assay (ELISA) can be applied. Some of these methods require the immobilization of the antibody on a surface and the titration of the target antigen to measure the dissociation constant KD and the association and dissociation rates ka and kd (also called on- and off rates kon and koff).

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Topics: Nano-CaptureLigands

How fluorescent proteins can be applied in SARS-CoV-2 research

Posted by Dr. Astrid Sitte on May 14, 2020 12:28:52 PM
Fluorescent proteins (FPs) like GFP or RFP and their derivatives are commonly used for virus research. Frequently, they are applied as fluorescent markers in microscopy or cell sorting experiments and as protein tags for protein purification, immunoprecipitation or protein-protein interaction assays. Here, we provide a short outline that describes how FPs have been used in the current SARS-CoV-2 research. Please note that this summary does not provide a complete overview; also, many referenced papers are pre-prints without peer reviews.
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Do you purify a special protein?

Posted by Dr. Astrid Sitte on Apr 24, 2020 9:33:54 AM

Why Spot-Tag can be used to purify any protein



Proteins often have very different individual characteristics and requirements for manipulation. Not every purification tag in combination with the desired purification resin is suitable for every protein. Some proteins are sensitive towards wash or elution buffers, while others are difficult to purify because the selected affinity resin can be contaminated by host cell proteins. Taking these points into consideration, the selection of both the affinity tag and the affinity resin can have a strong impact on protein purity and yield, which is often dependent on the target protein. Requirements for an affinity resin comprise (i) high purity (minimal contaminations), (ii) high yield of the purified protein, and (iii) a broad buffer stability. Ideally, affinity tags should be (iv) inert and (v) short in order to not or minimally affect the purified protein.

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Topics: Spot-Tag

Green fluorescent protein (GFP) in plant research

Posted by Dr. Astrid Sitte on Apr 1, 2020 5:02:51 PM
Although the jellyfish Aequorea Victoria Green fluorescent protein (GFP) was already discovered in the 1960s, it took three more decades until it was eventually cloned and could be utilized as a marker protein in E.coli and C. elegans. Since then it has developed into one of the most widely studied and exploited proteins in life sciences. Correspondingly, the importance of GFP was recognized in 2008 when the Nobel Committee awarded Osamu Shimomura, Marty Chalfie, and Roger Tsien the Chemistry Nobel Prize "for the discovery and development of the green fluorescent protein, GFP."
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Topics: GFP, A. Thaliana, plant research

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