Sample buffer

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A sample buffer (including loading buffer , English sample buffer, loading buffer ) indicated in biochemistry a solution with buffered pH , in front of a gel is added to the samples.

properties

A sample buffer fulfills various functions. The pH value of the sample is stabilized by the buffers it contains. Most of the time the same buffer system is used as in the electrophoresis buffer . Furthermore, sample buffers contain electrically neutral, water-soluble molecules of higher density such as. B. glycerol (30% v / v in a five-fold concentrated sample buffer), sucrose (40% w / v in a five-fold concentrated sample buffer) or Ficoll (20% w / v in a five-fold concentrated sample buffer) to increase the density of the sample, so that the samples sink into the pockets of the gel when the sample is applied . To identify the solvent front, anionic dyes are used in anionic electrophoresis , which due to their relatively small molecule size migrate faster than proteins or nucleic acids. With the optical control based on the moving colored band, the electrophoresis can be ended before the dye and thus also the samples have completely migrated through the gel and leave it again.

Proteins

Denaturation of a protein with SDS
Reduction of a disulfide bridge by DTT

In SDS-PAGE , bromophenol blue is usually used as the dye in the sample buffer (0.2 g / L final concentration). As a surfactant which is denaturing anionic surfactant sodium lauryl sulfate (SDS, 5 g / L final concentration) was used. In the case of NU-PAGE, BN-PAGE ( Blue native PAGE ) and isoelectric focusing , the protein dye Coomassie brilliant blue is occasionally used in the sample buffer , which is why phenol red is used instead of the bromophenol blue to better differentiate the solvent front. The buffer is composed analogously to the running buffer of SDS-PAGE with TRIS (250 mM final concentration), glycine and sodium lauryl sulfate. A reducing sample buffer also contains mercaptoethanol (5% V / V), dithiothreitol (10 mM) or dithioerythritol for the cleavage of disulfide bridges . Occasionally, with poorly soluble protein aggregates such as inclusion bodies , a nonionic chaotrope such as urea (8 molar) or thiourea (6 molar) is used in the sample buffer and in the gel. Since proteins have to be completely denatured before SDS-PAGE, the samples are mixed with sample buffer and then for 5 min. heated to 95 ° C.

In native gel electrophoresis , milder and nonionic surfactants such as digitonin , polysorbate (Tween) or octoxinol 9 (Triton X-100, Nonidet P-40), which are not as strongly denaturing, are used instead of sodium lauryl sulfate (SDS) . Octoxinols absorb UV radiation, which increases the background color with a fluorescent staining method and therefore interferes with the determination of the quantity. Occasionally the surfactants are left out in native gel electrophoresis, provided that the protein sought is sufficiently soluble. Furthermore, in native gel electrophoresis, reducing additives are not used to maintain the disulfide bridges. In a native gel electrophoresis, the sample is not heated to avoid denaturation.

In a CTAB-PAGE or BAC-PAGE , CTAB or 16-BAC are used as cationic surfactants in the sample buffer .

Nucleic acids

Agarose gel electrophoresis with three dyes in the sample buffer. The direction of migration is from the negative pole (connection to the black cable) to the positive pole (red cable). Migration in the picture from the bottom right (here are the wells in the gel into which the samples were applied) to the top left.

In agarose gel electrophoresis , bromophenol blue (running behavior comparable to around 400 bp DNA in a one percent gel and around 120 bp in a two percent gel), xylene cyanol blue (running behavior comparable to around 4000 bp DNA in a one percent gel and around 800 bp in a two percent gel), orange G. (Running behavior comparable to about 50 bp DNA in a one percent gel and less than 10 bp in a two percent gel), cresol red (running behavior comparable to about 1500 bp DNA in a one percent gel and about 300 bp in a two percent gel) or bromocresol green used as dyes, mostly in one concentration of 0.01% (m / V). In polyacrylamide gel electrophoresis , bromophenol blue and xylene cyanol blue run at 60 bp and 240 bp in six percent polyacrylamide gels and at 35 bp and 120 bp in ten percent polyacrylamide gels.

As a buffer in the sample buffer z. B. EDTA solution (50 mM in a five-times concentrated sample buffer), tris-EDTA buffer ( TE buffer ), TRIS- acetic acid -EDTA buffer ( TAE buffer ), TRIS- boric acid -EDTA buffer ( TBE buffer ) or lithium borate buffer is used. Using crystal violet in the sample buffer enables the DNA in the gel to be stained during gel electrophoresis without the need for subsequent irradiation with UV light in the case of subsequent gel extraction of the nucleic acids, provided that no TBE buffer, bromophenol blue or xylene cyanol blue is used. Bromophenol blue or xylene cyanol blue in combination with crystal violet lead to a poorer electrophoretic separation.

Some anionic dyes which do not interfere with the thermostable DNA polymerases in a polymerase chain reaction or a qPCR and in the latter case also with the fluorescence are e.g. B. quinoline yellow , xylene cyanol blue, brilliant blue , patent blue V , indigo carmine , red 2G , m- cresol purple , cresol red, neutral red , bromocresol green, acid violet 5 , bromophenol blue and orange G. These dyes can already appear in the PCR approach, which unites the pipetting process of sample and sample buffer before electrophoresis. Some of them are available in commercial PCR mixtures under brand names such as Red Taq , Green Taq , etc. A PCR buffer is used as the buffer for colored PCR mixtures.

literature

Individual evidence

  1. ↑ L. Ornstein, BJ Davis: Disc Electrophoresis -1. Background and Theory. In: Ann NY Acad Sci. Volume 121, 1964, pp. 321-349.
  2. ↑ Thierry Rabilloud: Two-Dimensional Electrophoresis Protocols. Chapter 2: Solubilization of proteins in 2DE: An outline. Humana Springer, 2009, ISBN 978-1-58829-937-6 . (PDF) .
  3. ↑ Calbiochem Booklet: A Guide To The Properties And Uses Of Detergents. 2001. (PDF; 597 kB) .
  4. ^ A b c Lela Buckingham: Molecular Diagnostics. FA Davis, 2011, ISBN 978-0-803-62975-2 . P. 98
  5. ↑ Free Patents Online US patent application 20120219957 . Retrieved December 2, 2013.