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64 Winkler and Campbell<br />
3. For incubation of the membrane with the biotinylated protein sample and streptavidin,<br />
follow the instructions of steps 1–4 in Subheading 3.3.1. with the exception<br />
of using the HRP-labeled streptavidin at a concentration of 0.1 �g/mL instead of<br />
the HRP-labeled proteins (antibodies).<br />
4. Detection: Follow step 6 of Subheading 3.3.1. or step 2 of Subheading 3.3.2.<br />
4. Notes<br />
1. Reagents must be protected from moisture. To avoid condensation, reagent<br />
bottles stored in the fridge or freezer should be unopened and warmed up to room<br />
temperature for approx 30 min before use.<br />
2. If the binding is expected to be very weak the use of PBS, TBS, PBS-T, or TBS-T<br />
instead of blocking buffer is possible. In that case, due to possible unspecific<br />
binding, a higher background signal during detection may occur.<br />
3. To avoid problems due to formation of poor solubility in the urea, mix the in situ<br />
activated solutions for about 30 min. Then centrifuge the mixtures and transfer<br />
the supernatant to vials for the synthesis.<br />
4. Because of the flexibility and linear structure of the molecule, �-alanine is<br />
commonly used for modification of the cellulose as well as a spacer to achieve a<br />
higher distance of the amino functions from the cellulose matrix. However, other<br />
functionalizations are also possible (65).<br />
5. The membrane can be stored at −80oC for several months until needed. For<br />
longer storage a slight loss of loading is possible. Storage of the membranes at<br />
−80◦C is recommended. The membranes can also be stored at −20◦Cor+4◦C, but there is a higher risk of losing activity over a long storage time.<br />
6. Do not stain the spots too strongly because if the the amount of absorbed<br />
bromophenol blue is too high, some of this dye could be incorporated in the<br />
peptide. In that case removal of the color is almost impossible and could affect<br />
the detection. Because bromophenol blue has a blue color in a basic environment,<br />
e.g., primary amino functions, a difference in the intensity of the color of the<br />
spots is normal due to different acidity of the coupled amino acids.<br />
7. To cover the whole area of large spots, it is recommended to use at least 20%<br />
more amino acid solution volume than in step 5 of Subheading 3.1.<br />
8. Release of peptides with ammonia might work only on homemade membranes.<br />
Commercially available membranes with amino functional groups often have<br />
other than ester bonds between the spacer and cellulose. For such membranes<br />
the use of additional linkers might be necessary (e.g., thioester (66), HMB linker<br />
(67), Rink linker (68)).<br />
9. To minimize possible contamination with side products from the edge of the spot,<br />
the diameter of the punched-out discs should be smaller than the spot diameter.<br />
10. In general, the peptide sequences should be between 6 and 15 amino acids in<br />
length; spot syntheses of longer (e.g., 34 and 44 amino acids (69,70)) and shorter<br />
(e.g., 2 amino acids (71)) peptides are also described. With shorter peptides, the<br />
measurable activity might be too low. For the synthesis of long peptides, the