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Dr. Astrid Sitte

Recent Posts

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

 

Introduction

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

Virus research using GFP

Posted by Dr. Astrid Sitte on Mar 26, 2020 1:05:30 PM

An extract of the published literature since 2016

This blog provides references from the last 4 years in virus research using GFP. Most publications report how immunoprecipitation (IP)/Co-IP of GFP-fusions was conducted to identify host cell binding partners of virus proteins. In addition, mass spectrometry analysis and functional assays have been performed.

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Topics: GFP

What is antibody specificity and affinity? And why is it important for immunoprecipitation?

Posted by Dr. Astrid Sitte on Mar 12, 2020 5:17:58 PM

Introduction of Affinity vs. Specificity

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Topics: Immunoprecipitation

How to obtain a low background in immunoprecipitation assays

Posted by Dr. Astrid Sitte on Mar 10, 2020 2:02:57 PM

Introduction

A high background from unspecific binding of proteins is a common problem in immunoprecipitation (IP). In this blog, the origin of background and different optimization strategies are discussed. Furthermore, it is shown how you can achieve a low background when using the ChromoTek GFP-Trap®, a ready to use reagent for IP of a GFP-tagged proteins.

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Topics: GFP-Trap, Immunoprecipitation

Immunoprecipitation without additional bands

Posted by Dr. Astrid Sitte on Mar 6, 2020 3:03:09 PM

Estimated reading time:  3 minutes

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Topics: Immunoprecipitation

The importance of blocking when using Nano-Secondaries for IF

Posted by Dr. Astrid Sitte on Jan 28, 2020 5:04:30 PM

Introduction

Blocking is an essential step during the preparation of a sample for immunofluorescence detection. Blocking improves the sensitivity by reducing nonspecific background and therefore increases image quality. Insufficient blocking results in higher background noise and over-blocking can even mask the specific signal.

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Topics: Nano-Secondaries, staining

What GFP-Trap should I use for my immunoprecipitation?

Posted by Dr. Astrid Sitte on Jan 20, 2020 4:42:38 PM

ChromoTek GFP-Trap® is optimized for the immunoprecipitation of GFP-tagged proteins and their interacting factors.

The GFP-Trap consists of the ChromoTek anti-GFP Nanobody/ VHH that is coupled to 3 different types of beads or is immobilized in a 96 multiwell plate:

  • Agarose beads
  • Magnetic Agarose beads
  • Dynabeads™
  • 96 Multiwell Plate

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

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

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Topics: GFP-Trap, GFP Immunoprecipitation, GFP Nanobody, Dynabeads

How to overcome the limitations of antibody-based affinity resins for protein purification

Posted by Dr. Astrid Sitte on Dec 18, 2019 2:19:42 PM

Introduction

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Topics: affinity resin, affinity purification, Spot-Tag, Spot Nanobody, protein purification, epitope tag

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