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Dr. Astrid Sitte

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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
  • Magnetic Particles M-270
  • 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

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

What is one-step immunostaining?

Posted by Dr. Astrid Sitte on Jul 26, 2019 2:45:28 PM

Simultaneous immunostaining, also called one-step immunostaining vs. sequential immunostaining. Nano-Secondaries stain different primary antibodies equally well in one-step staining and sequential staining.

HeLa cells were immunostained with different primary antibodies and Nano-Secondaries Alexa Fluor® 647 (1:1,000, magenta). Cell nuclei were stained with DAPI (blue). Scale bar, 20 μm.

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Topics: live cell imaging, Secondary antibody, Nano-Secondaries, One-step immunostaining, staining

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