Protein tools molecular weight

Contact Sales. Notice to purchaser Our products are to be used for Research Use Only. Documents Components Image Data. Your partner for molecular diagnostic development and supply As an experienced, reliable solutions provider, we offer full-scale customization services for a vast array of reagents and instruments backed by a quality management system, on-time delivery, and dependable support—giving you a competitive advantage in the ever-changing clinical landscape.

Stem cell services Clinical-grade stem cell services Research-grade stem cell services Outsourcing stem cell-based disease model development Gene and cell therapy manufacturing services Services Facilities Our process Resources. Partner with Takara Bio! Molecular diagnostics Applications Solutions Partnering Webinar: Speeding up diagnostic development Contact us Vaccine development Characterizing the viral genome and host response Identifying and cloning vaccine targets Expressing and purifying vaccine targets Immunizing mice and optimizing vaccine targets.

Log in to enjoy additional benefits Want to save this information? An account with takarabio. If the unknown protein is elongated, it can easily elute at a position twice the molecular weight of a globular protein. The gel filtration column actually separates proteins not on their molecular weight but on their frictional coefficient.

Since the frictional coefficient, f , is not an intuitive parameter, it is usually replaced by the Stokes radius R s. R s is defined as the radius of a smooth sphere that would have the actual f of the protein. This is much more intuitive since it allows one to imagine a real sphere approximately the size of the protein, or somewhat larger if the protein is elongated and has bound water.

As mentioned above for Eq. The Stokes radius R s is larger than R min because it is the radius of a smooth sphere whose f would match the actual f of the protein. It accounts for both the asymmetry of the protein and the shell of bound water. Siegel and Monte [ 4 ] argued convincingly that the elution of proteins from a gel filtration column correlates closely with the Stokes radius, R s , presenting experimental data from a wide range of globular and elongated proteins.

BsSMC eluted in fraction In repeated experiments, the average R s was The standard proteins should span R s values above and below that of the protein of interest but in the case of SMC protein from B.

The literature generally recommends determining the void and included volumes of the column and plotting a partition coefficient K AV [ 4 ]. However, we have found it generally satisfactory to simply plot elution position vs R s for the standard proteins. This generally gives an approximately linear plot, but otherwise, it is satisfactory to draw lines between the points and read the R s of the protein of interest from its elution position on this standard curve.

A gel filtration column can determine R s relative to the R s of the standard calibration proteins. The R s of these standards was generally determined from experimentally measured diffusion coefficients.

Some tabulations of hydrodynamic data list the diffusion coefficient, D, rather than R s , so it is worth knowing the relationship:. Typical proteins have D in the range of 10 -6 to 10 -7 cm 2 s However, if one determines both R s and S , this permits a direct determination of molecular weight, which cannot be deduced from either one alone.

This is described in the next section. With the completion of multiple genomes and increasingly good annotation, the primary sequence of almost any protein can be found in the databases. The molecular weight of every protein subunit is therefore known from its sequence. But an experimental measure is still needed to determine if the native protein in solution is a monomer, dimer, or oligomer, or if it forms a complex with other proteins.

If one has a purified protein, the molecular weight can be determined quite accurately by sedimentation equilibrium in the analytical ultracentrifuge. This technique has made a strong comeback with the introduction of the Beckman XL-A analytical ultracentrifuge.

There are a number of good reviews [ 14 , 15 ], and the documentation and programs that come with the centrifuge are very instructive. What if one does not have an XL-A centrifuge or the protein of interest is not purified? In , Siegel and Monte [ 4 ] proposed a method that achieves the results of sedimentation equilibrium, with two enormous advantages. First, it requires only a preparative ultracentrifuge for sucrose or glycerol gradient sedimentation and a gel filtration column.

This equipment is available in most biochemistry laboratories. Second, the protein of interest need not be purified; one needs only an activity or an antibody to locate it in the fractions. This is a very powerful technique and should be in the repertoire of every protein biochemist.

The methodology is very simple. The protein is run over a calibrated gel filtration column to determine R s and hence f. Separately, the protein is centrifuged through a glycerol or sucrose gradient to determine S. One then uses the Svedberg equation Eq. This is pretty simple! This is more than enough precision to distinguish between monomer, dimer, or trimer. Since the early s, electron microscopy has become a powerful technique for determining the size and shape of single protein molecules, especially ones larger than kDa.

Two techniques available in most EM laboratories, rotary shadowing and negative stain, can be used for imaging single molecules. Cryo-EM is becoming a powerful tool for protein structural analysis, but it requires special equipment and expertise. For a large number of applications, rotary shadowing and negative stain provide the essential structural information.

For rotary shadowing, a dilute solution of protein is sprayed on mica, the liquid is evaporated in a high vacuum, and platinum metal is evaporated onto the mica at a shallow angle. The mica is rotated during this process, so the platinum builds up on all sides of the protein molecules. The first EM images of single protein molecules were obtained by Hall and Slayter using rotary shadowing [ 16 ]. Their images of fibrinogen showed a distinctive trinodular rod.

However, rotary shadowing fell into disfavor because the images were difficult to reproduce. Protein tended to aggregate and collect salt, rather than spread as single molecules. For reasons that are still not understood, the glycerol greatly helps the spreading of the protein as single molecules. Pullman never published his protocol, but two labs saw his mimeographed notes and tested out the effect of glycerol, as a part of their own attempts to improve rotary shadowing [ 17 , 18 ].

They obtained reproducible and compelling images of fibrinogen the first since the original Hall and Slayter study and confirming the trinodular rod structure and spectrin the first ever images of this large protein. The technique has since been used in characterizing hundreds of protein molecules. SMC protein from B. The fibrinogen molecules show the trinodular rod, but these images also resolved a small fourth nodule next to the central nodule [ 20 ], not seen in earlier studies.

The central nodule is about 50 kDa, and the smaller fourth nodule is about 20 kDa. The "hexabrachion" tenascin molecule [ 21 ] illustrates the power of rotary shadowing at two extremes. First, the molecule is huge. At the larger scale, the EM shows that each arm is an extended structure, matching the length expected if the repeating domains are an extended string of beads. At the finer scale, the EM can distinguish the different sized domains. The inner segment of each arm is a string of 3.

The terminal knob is a single kDa fibrinogen domain. The R min of these domains are 0. Rotary shadowing EM can visualize single globular domains as small as 10 kDa 3. Negative stain is another EM technique capable of imaging single protein molecules. It is especially useful for imaging larger molecules with a complex internal structure, which appear only as a large blob in rotary shadowing. Importantly, noncovalent protein—protein bonds are sometimes disrupted in the rotary shadowing technique [ 8 ], but uranyl acetate, in addition to providing high resolution contrast, fixes oligomeric protein structures in a few milliseconds [ 22 ].

An excellent review of modern techniques of negative staining, with comparison to cryo-EM, is given in [ 23 ]. The simple picture of the molecule produced by EM is frequently the most straightforward and satisfying structural analysis at the 1—2-nm resolution. When the structure is confirmed by hydrodynamic analysis, the interpretation is even more compelling. Similar hydrodynamic analysis can be used to analyze multisubunit protein complexes.

The protein complex called DASH or Dam1 is involved in attaching chromosomal kinetochores to microtubules in yeast. These complexes further assemble into rings that can form a sliding washer on the microtubule [ 24 , 25 ].

The basic ten-subunit complex has been purified from yeast and has also been expressed in Escherichia coli and purified this required the heroic effort of expressing all ten proteins simultaneously [ 24 ]. Gel filtration is shown in a and sucrose gradient sedimentation in b. Independent calibration curves using standard proteins are shown in black and green. Dark and light blue show Spc34p in yeast cytoplasmic extract and in the purified recombinant protein.

The enzyme cascade provides substrate specificity and activation, conjugation, and ligation steps. Proteins may be mono-ubiquitinated, or additional ubiquitin molecules may bind to the initial ubiquitin molecule, causing poly-ubiquitination.

Ubiquitination can mark proteins for degradation and is also important for cellular signaling, the internalisation of membrane proteins, and the development and regulation of transcription.

Ubiquitin can be removed from proteins by deubiquitinating enzymes, which then lowers the molecular weight Figure 4. As Western blotting SDS-PAGE is performed in denaturing conditions, most protein complexes that are composed of proteins linked via non-covalent bonds disassociate during sample preparation and electrophoresis, with the component proteins then running as monomers. In these cases the observed molecular weight can be substantially higher than the predicted, calculated monomeric form Figure 5.

Some proteins, especially transmembrane proteins and proteins with hydrophobic domains, can aggregate during cell lysis as they are released from their native protein complexes and lipid membranes. These aggregates have high molecular weights and may not represent interactions that occur in their native states.

Many proteins encoded by a single gene exist in more than one sequence variant, or protein isoform, due to alternative splicing during mRNA maturation.

This assay utilizes a thioflavin T fluorescent stain to provide a qualitative and quantitative measure of protein misfolding within cells.

The functionality of this method was first assessed in renal proximal tubule epithelial cells treated with various endoplasmic reticulum ER stress inducers. Once established in the renal model system, we analyzed the ability of some known chemical chaperones to reduce ER stress.