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The Protein Man's Blog | A Discussion of Protein Research

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Magnetic Beads for Immunoprecipitation

Magnetic Beads

Researchers have traditionally used agarose beads for immunoprecipitation (IP). However, there has been a growing trend in recent years favoring the use of magnetic beads. According to a recent survey of 1,013 scientists, 60% of the respondents will start using magnetic technology in the next three years.

PVDF or Nitrocellulose - Which Membrane is Best?

PVDF or Nitrocellulose

When it comes to Western blotting, choosing the right membrane for your application usually spells the difference between success and failure. However, this is usually easier said than done since you need to consider the properties of your protein and the downstream detection steps required in your application before you can determine which membrane can give you the results that you need.

A Look into the 6XHis Tag and its Uses

His6x tag

The 6xHis tag, also known as polyhistidine tag, His6 tag and/or hexa histidine tag, is an amino acid motif consisting of at least 6 histidine residues fused to the carboxyl (C-) or amino (N-) terminus of a target protein in transfected cells. This tag is most commonly used in the production of recombinant proteins since the string of histidine residues binds to several types of immobilized ions (such as nickel, cobalt and copper) under specific buffer conditions to allow for the simple detection and purification of His-tagged proteins.

Which agarose (sepharose) to choose? 2, 4 or 6%? Crosslinked?

Protein Purification

Agarose beads are small spherical beads of agarose gel which are commonly used in gel filtration or molecular size exclusion chromatography and biomolecular purification and immobilization. These beads act as porous gel to filter mixtures of molecules based on their individual sizes. And since these beads are easy to activate, they can also be used to bind biomolecules in a reversible or irreversible manner. In addition, their inert nature and unique internal surface area can also be activated for ligand attachment, making them the ideal basis for various affinity chromatography beads such as protein A and G, and glutathione.

Spectrophotometry and Its Application in Protein Estimation

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Basically, spectrophotometry is one of the most widely used analytical procedures in biochemistry. It is commonly used to estimate the level of an analyte in solution and is ideal for simple routine determination of small quantities of materials. This method is based on the two laws of light absorption by solutions, namely Lambert's Law and Beer's Law.

Using Tags in Protein Purification: When Should You Use Them?

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Tagging your protein of interest can be extremely useful since it simplifies the purification protocol, improves the yield and solubility of your protein of interest and promotes the proper folding of their fusion partners. Due to their versatility, affinity tags (peptide sequences that are appended to the target protein) are recognized as one of the most powerful tools that can be used for basic biological research and in structural and functional proteomics as well.

How Proteins Interact with DNA and RNA to Influence Nucleic Acid

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Due to the fact that nucleic acids carry genetic information and that proteins regulate various life processes, they are considered to be two of the most important biomolecules in any living organism. In addition, their interactions play a crucial role in most biological processes, which include everything from replication,transcription and recombination to enzymatic eventsusing nucleic acids as substrates. Taking all of these things into consideration, it is not surprising why protein-nucleic acids interactions have been the subject of intensive research for the past few years.

What You Need to Know About NTA and IDA Ligands

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Since 1975, immobilized metal affinity chromatography (IMAC) has been popularly used in purifying proteins, especially those that are fused to a polyhistidine tag, typically a 6X His tag. This process gained immense popularity since it allows for the efficient purification of proteins, even those from crude lysates. In addition, its robust nature makes it ideal for methods that require protein-specific conditions. Its functional simplicity, affordability and compatibility with a wide range of reagents also add to its popularity.

A Guide to Protein Cross Linkers


Protein crosslinking reagents or crosslinkers can be accurately defined as molecules containing two or more reactive ends that are capable of chemically attaching to specific functional groups on proteins or other molecules. These reagents are generally used in creating detectable scientific probes to facilitate a number of proteomics methods, including Western blotting, ELISA and other strategies for studying protein-protein interactions.


Interfering Agents of 2D Electrophoresis and How to Remove Them

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Most biological samples contain non-protein substances and/or contaminants that may interfere with the resolution of the electrophoretic separation. Considering the fact that cell lysates usually contain macromolecules and small ionic molecules which may interfere with the electrophoretic process, these agents should be eliminated prior to electrophoresis, especially if their amount exceeds the critical interference threshold.

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