Staining blots

Fleck, m. spot speck, stain, blot, flaw, patch ... 

CBB G-250 is another popular total protein stain. Researchers blotting 2-D PAGE gels particularly favor it because it is compatible with mass spectrometry. Stained blots provide good media for archiving 2-D PAGE separations. A version of SYPRO Ruby, formulated for blots, is a very sensitive total protein stain. 

If the IPG gels are not used for a second dimension run, the gels are further processed (stained, blotted) as described for SDS gels. 

Stained blots are dried on air on a sheet of filter paper. If radioactive compounds are also transferred, autoradiography of the dry membrane is possible at -70 °C. 

Acrylamide in the unpolymerized form is a skin irritant and a potential neurotoxin. Wear gloves and a mask while weighing the dry powder. Do not breathe the dust. Prepare all acrylamide solutions in the hood. Do not mouth pipet any solutions used for gel formation, staining, blotting, or detection. 

Fig. I. Examples of Western blots stained with colloidal gold for total protein followed by immunostaining of individual antigensJ.anes 1—3 Proteins on a Western blot from a cytoplasmic extract of poliovirus-infected HEp-2 cells were stained with colloidal gold. The probing monoclonal antibodies, recognizing the viral proteins VP1 and precursor, VPO and VP2, and VP3, respectively, are detected by peroxidase-coupled rabbit-antimouse antibody (asterisks). Lane 4 Western blot of an E. coli lysate, containing a fusion protein composed of protein A and the poliovirus protein 2B. The fusion protein (arrowhead) is detected on the gold-stained blot by peroxidase-coupled IgG that binds to the protein A moiety.

Trays for staining, blotting, and incubation III. EXPERIMENTAL PROCEDURE... 

In some cases, stained blots are used only to identify protein band patterns while leaving the gel unmodified for subsequent steps (UNITB3.3). If such minimal protein transfer is desired, contact blotting is a suitable alternative. This unit also describes procedures for eluting proteins from membranes using detergents (Basic Protocol 2) or acidic extraction with organic solvents (Alternate Protocol 4). 

Fig, 1. Left Western blot stained with HCP polyclonal antibodies. Lane 1 contains mw markers (20, 30, 40, 50, 60, 80, 120, 220 kD), lane 2 is a sample of production fraction 2.1 diluted 250 times, lane 3 contains a sample of production fraction 3.1 diluted 10 times, lane 4 contains the HCP-mix diluted 10 times, lane 5 contains an undiluted sample of production fraction 5.1. Lane 6 contains normal rabbit serum as a negative control. Right, residual protein staining blot (right, lane 1 mw markers, lane 2 empty, lanes 3-9 correspond with lanes 1-6 of the western blot)... 

Fig. 2. Depletion of Mcl-l protein levels by antisense treatment. Neutrophils were reversibly permeabilised with streptolysin O and incubated with 20 iJuM carboxyfluorescein (CF), inverse antisense (Inverse) or antisense sequence 1 (AS) of chimeric oligodeoxynu-cleotides. Suspensions were incubated in either the absence (—) or presence (+) of GM-CSF. Western blots were analysed on aliquots of 10 cells, 4 h after permeabilisation. Actin (Ponceau S-stained blots) is shown to indicate equivalence of loading. Representative blot of 3 independent experiments. Antisense sequences and structures are given by Moulding et al. [28]. 

Method Sensitivity (Threshoids in ng/O.Scm Bands) Time Variability of the Stain Blotting Possible After Staining ... 

Transfer to three successive drops of stain, blotting excess liquid but not drying between drops. 

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