Capillary blots

Blot the proteins onto the affinity membrane either by dot blot (Protocol 4.8), by electrotransfer (Protocol 2.5.3), or by capillary blotting (handling analogously to Southern blotting Protocol 2.5.9). 

In both Southern and Northern analyses restriction-digested DNA fragments, mRNA, and polyA mRNA are separated by size when electrophoresed on agarose gel. The separated molecules are transferred, by electroblotting or capillary blotting, on to a nylon or nitrocellulose membrane. The immobilized RNA or DNA is reacted with a radiolabeled, chemiluminescent, or fluorescent probe that is complementary to the DNA/RNA of interest, unbound probe is washed off, and the membrane exposed, in the case of radioactive probes, to radioautographic film to visualize the sample of interest. 

For ease of handling the DNA fragments are transferred from the gel to a solid support matrix by capillary blotting, thus maintaining their relative positions. 

Gels can also be transferred by capillary blotting by a slight modification of the procedure described in Chapter 7. Pretreat the gel with denaturing solution as described above (Section 3.3., step 1) and prewet the membrane (Section 3.3., step 5), then carry out steps 7-12 of the Methods section of Chapter 7, but using 20X SSC as the transfer buffer in place of lOX SSC. Continue from the rinsing step above (Section 3.3., step 16). 

HaargefaC capillary air bleed/ boiling capillary/ air leak tube dist Siedekapillare capillary blotting Diffusionsblotting capillary breaking/ capillary fracture (fibers) Kapillarbruch... 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

When the adjustment has been completed, rinse the capillary well with a stream of distilled water from a wash bottle and then dry by blotting with filter paper. Insert the capillary through an inverted cone of quantitative filter paper and clamp vertically over a small beaker. Lower the levelling bulb until the mercury drops just cease to flow,... 

The transport of many compounds takes place through interstices of polymer chains filled with aqueous medium [52], In such cases, the rate of mass transport is directly proportional to the degree of hydration of the membranes [53]. The most widely accepted method for determining the hydration of membranes is to equilibrate the membranes in water or buffer and weigh these membranes after blotting [54], In a newer method, the matrices to be studied are placed on a sintered glass funnel which is attached to a capillary filled with water. The absorption of water results in the movement of the capillary front [55],... 

C2. Chomczynski, P., One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201, 134-139 (1992). 

The efficiency of reagent removal from the capillary gap was assessed by filling capillary gaps between blank slides with peroxidase-streptavidin complex. After picking up the solution, the slide pairs were blotted for 1 min. The slide pairs were then subjected to three changes in buffer, followed by three, 1-min... 

FIGURE 3.8 Setup for a Southern blot. The agarose gel containing the separated DNA is placed in contact with a nitrocellulose sheet, then pressed with filter paper and paper towels. The buffer in the wet gel moves by capillary action into the filter paper and towels the lower reservoir of buffer helps promote the migration of the DNA from the gel onto the nitrocellulose sheet. 

The actual blotting process may be accomplished by one of two methods passive (or capillary) transfer and electroblotting. In passive transfer, the membrane is placed in direct contact with the polyacrylamide gel and organized in a sandwich-like arrangement consisting of (from bottom to top) filter paper soaked with transfer buffer, gel, membrane, and more filter paper. The sandwich is compressed by a heavy weight. Buffer passes by capillary ac-... 

Both Parts I and II have been completely rewritten and reflect the many advances in biochemistry-molecular biology theory and techniques. Especially noteworthy have been the technical advances in chromatography (perfusion, FPLC, bioaffinity), electrophoresis (pulsed gel, capillary, nucleic acid sequencing), spectrophotometry (nmr, ms, and diode array detectors), and molecular biology (microsequencing of proteins and nucleic acids, blotting, restriction enzymes). 

When a penetrating colored dye solution is injected into a package it detects channels or voids in the sealed area via capillary action and pinholes in nonporous materials via blotting on a paper tissue. Packs with at least one transparent component are more suitable for viewing the results. Dye penetration is more difficult to use on packages of porous materials, such as paper. 

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