Separation of proteins by SDS-PAGE

An advance in the SDS-PAGE separation of proteins has been the optimization of the mini gel system. Mini gel systems are available from a number of 

CHARACTERIZATION OF CELLULAR ANTIGENS Separation of proteins by PAGE 

Incubation with specific primary monoclonal antibody  

Incubation with secondary antibody conjugated to marker  

10% (w/v) ammonium persulfate Water-saturated isobutanol 2 X SDS-PAGE sample buffen 80 mM Tris-HCl pH 6.8.50 mM DTT, 2% (w/v) SDS, 10% (v/v) glyceroL 0.01% (w/v) bromophenol blue SDS-PAGE nmniiig buffer 192 mM tydne, 25 mMTris, 0.1% (w/ SDS 

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Figure 7.1 Separation of proteins by SDS-PAGE. Protein samples are incubated with SDS (as well as reducing agents, which disrupt disulfide linkages). The electric field is applied across the gel after the protein samples to be analysed are loaded into the gel wells. The rate of protein migration towards the anode is dependent upon protein size. After electrophoresis is complete, individual protein bands may be visualized by staining with a protein-binding dye (a). If one well is loaded with a mixture of proteins, each of known molecular mass, a standard curve relating distance migrated to molecular mass can be constructed (b). This allows estimation of the molecular mass of the purified protein...

To remove the nonbound dye we precipitated the protein using the chloroform/ methanol method. The pellet was briefly dried and then dissolved at a protein concentration of 0.5-1.0 mg/ml in a buffer containing 8.0 M urea and 20 mM sodium phosphate (pH 8.0), and stored at -70°C. Protein labeling efficiency was determined after separation of proteins by SDS-PAGE by UV illumination (280 nm) of nonstained gels. The stoichiometry of labeling with 5-IAF (moles of dye per moles of protein) was determined according to Simon and Taylor (1986). 

Figure 3.4 Western blotting technique. (A) Size separation of proteins by SDS-PAGE. (B) Transfer of the separated proteins onto nitrocellulose (NC) or poly-vinylidine fluoride (PVDF) membrane by electro blotting. (C) Visualization of proteins of interest by specific enzyme-conjugated antibody using immuno-staining. (D) Visualization of whole-protein extract by Coomassie Blue staining (left side), and immunostaining (right side), respectively.

Foote and coworkers [120] developed a microfabricated system with the ability to electrophoretically preconcentrate fluorescently labeled proteins prior to their separation (see Fig. 6). The authors were able to preconcentrate the proteins using a porous silica membrane situated between adjacent microchannels that allowed for the passage of buffer ions, but excluded larger migrating molecules, such as proteins. Preconcentration factors of 600-fold were achieved using this on-chip format followed by an electrophoretic separation of proteins with SDS-PAGE. Using this chip, fluorescently labeled ovalbumin was detected at concentrations as low as 100 fmol by a combination of field-amplified injection and preconcentration at the membrane prior to microchip electrophoresis. 

Separation of polypeptides by SDS-PAGE on mini gels 272 Electrotransfer of proteins from gels to nitrocellulose membranes 275 Staining membranes with Ponceau S after transfer to obtain the total protein pattern 276 Inununoblotting 278... 

An elegant variant has been described by Muilerman et al. (1982). They were faced with the problem to identify a particular enzyme after it had been denatured and separated from other proteins by SDS-PAGE. After transferring the denatured proteins to nitrocellulose membranes and quenching of the blot, the membrane was over-... 

Rat liver membranes presumed to be from Golgi and lysosomal structure were isolated by immunoadsorption on Dynabeads M-500 (Dynal) conjugated to, respectively, anti-API and anti-LAMPl antibodies. After separation of membrane proteins by SDS-PAGE, endogenous ARDl was detected by immunoblotting with specific polyclonal anti-ARDl antibodies (Vitale et al, 1998a). 

Figure 52-3. Diagrammatic representation of the major proteins of the membrane of the human red blood cell separated by SDS-PAGE. The bands detected by staining with Coomassie blue are shown in the two left-hand channels, and the glycoproteins detected by staining with periodic acid-Schiff (PAS) reagent are shown in the right-hand channel. (Reproduced, with permission, from Beck WS, Tepper Ri Hemolytic anemias iii membrane disorders, in Hematology, 5th ed. Beck WS [editor]. The MiT Press, 1991.)...

Separation of the purified PL1, PL2 and PL3 isoenzymes by SDS-PAGE yielded single protein bands that corresponded to a molecular mass of 42 kDa (6), 4 kDa higher than calculated from the deduced amino acid sequences. Isoelectric focusing revealed a pi of >10 for each isoenzyme similar to that of the basic Ech PLs (15). 

Poehling reported a microscale two-dimensional gel electrophoresis system 25 years ago (Poehling and Neuhogg, 1980 Neuhoff, 2000). In that system, separation in the IEF dimension was performed in thin, gel-filled tubes. After IEF, the gel was transferred to a 3 cm x 3.5 cm polyacrylamide gel, where proteins were separated by SDS-PAGE. Several hundred components were resolved from a few micrograms of protein homogenate. 

Recently, Vasilescu et al. demonstrated the use of formaldehyde to preserve protein interactions in vivo followed by immunoaffinity purification of a targeted complex, cross-link reversal via heating at 95°C, separation by SDS-PAGE, and identification of bands by LC-MS/MS.7 Tagwerker et al. utilized formaldehyde cross-linking in conjunction with a novel tag-based affinity purification method.36... 

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