Transfer Membranes

More documents

Recommendations

Info

AppliChem Transfer Membranes by Application Application Membrane Catalog Number Alkaline Reprobe Nylon Positively Charged A5255 Blotting Amino Acid Pure Nitrocellulose Unsupported A5250 or A5239 Analysis PVDF-Star A5243 Biotinylated Pure Nylon Neutral A4399 or A5248 Probes Reprobe Nitrocellulose Supported A5237 or A5242 Cell Blotting Reprobe Nylon Positively Charged A5255 Pure Nylon Neutral A4399 or A5248 Cell Labeling Pure Nylon Neutral A4399 or A5248 Chemilumines- Pure Nylon Neutral A4399 or A5248 cent Probes Reprobe Nitrocellulose Supported A5237 or A5242 Chromogenic Pure Nitrocellulose Unsupported A5250 or A5239 Reprobe Nitrocellulose Supported A5237 or A5242 Colony Lift Pure Nylon Neutral A4399 or A5248 Reprobe Nitrocellulose Supported gridded discs * DNA Reprobe Nylon Positively Charged A5255 Hybridization Pure Nylon Neutral A4399 or A5248 Reprobe Nitrocellulose Supported A5237 or A5242 Dot/Slot Reprobe Nylon Positively Charged A5255 Blotting Reprobe Nitrocellulose Supported A5237 or A5242 Drug Binding Pure Nitrocellulose Unsupported A5250 or A5239 Assay Reprobe Nitrocellulose Supported A5237 or A5242 Hormone Pure Nitrocellulose Unsupported A5250 or A5239 Binding Reprobe Nitrocellulose Supported A5237 or A5242 Immuno- Pure Nitrocellulose Unsupported A5250 or A5239 blotting PVDF-Star A5243 Library Pure Nylon Neutral A4399 or A5248 Screening Reprobe Nitrocellulose Supported A5237 or A5242 Multiple Reprobe Nylon Positively Charged A5255 Reprobings Application Membrane Catalog Number Northern Reprobe Nylon Positively Charged A5255 Transfers Pure Nylon Neutral A4399 or A5248 Reprobe Nitrocellulose Supported A5237 or A5242 Pure Nitrocellulose Unsupported A5250 or A5239 Non-Radio- Pure Nylon Neutral A4399 or A5248 active Probes Reprobe Nitrocellulose Supported A5237 or A5242 Nucleic Acid Reprobe Nylon Positively Charged A5255 Transfer Pure Nylon Neutral A4399 or A5248 Reprobe Nitrocellulose Supported A5237 or A5242 Pure Nitrocellulose Unsupported A5250 or A5239 Plaque Lifts Reprobe Nitrocellulose Supported gridded discs * Protein Pure Nitrocellulose Unsupported A5250 or A5239 Blotting PVDF-Star A5243 Protein Pure Nitrocellulose Unsupported A5250 or A5239 Precipitation Reprobe Nitrocellulose Supported A5237 or A5242 Protein PVDF-Star 0.1 µm pore size * Sequencing Protein Pure Nitrocellulose Unsupported A5250 or A5239 Transfer PVDF-Star A5243 Radiolabeled Reprobe Nylon Positively Charged Probe A5255 Reprobe Nitrocellulose Supported A5237 or A5242 RNA Reprobe Nylon Positively Charged A5255 Hybridizations Reprobe Nitrocellulose Supported A5237 or A5242 Solid Phase PVDF-Star A5243 Assay Southern Reprobe Nylon Positively Charged A5255 Transfers Reprobe Nitrocellulose Supported A5237 or A5242 Pure Nylon Neutral A4399 or A5248 UV Pure Nylon Neutral A4399 or A5248 Crosslinking Vacuum Reprobe Nylon Positively Charged A5255 Blotting Reprobe Nitrocellulose Supported A5237 or A5242 Westerns Pure Nitrocellulose Unsupported A5250 or A5239 PVDF-Star A5243 * available on request applications Transfer Membranes • AppliChem © 2008
s e l e c t i o n Selecting a Transfer Membrane Selecting the appropriate membrane is critical to the success of a nucleic acid or protein transfer procedure. AppliChem supplies several types of membranes for hybridization technology, each exhibiting different performance characteristics which can directly affect the outcome of a specific technique. Below are some of the more frequently performed procedures and features of hybridization membranes. Rehybridizations AppliChem offers membranes recommended for rehybridization procedures: Pure Nylon Neutral, Reprobe Nylon Positively Charged and Reprobe Nitrocellulose Supported, a supported pure nitrocellulose. Reprobe Nylon Positively Charged membranes can be most frequently reprobed. On nylon membranes, the number of reprobing steps is a function of the amount of hydrolysis to which the membrane is exposed during the protocol, and the additive effects of hot water, sodium hydroxide and an acidic environment. Sodium hydroxide solutions deteriorate the nylon matrix and are not recommended in procedures where reprobing steps are required. The polyester support web used in manufacturing Reprobe Nitrocellulose Supported allows the membrane to be reprobed several times. Because the binding capacity of nitrocellulose is less than that of nylon (100 µg/cm 2 vs. 400 µg/cm 2 ), the potential number of rehybridizations is fewer. See below for more details (Southern (DNA) and Northern (RNA) Hybridizations) Non-Radioactive Probes Reprobe Nylon Positively Charged may be used particularily where multiple probing is involved. UV Crosslinking For covalent binding of nucleic acids to a transfer membrane, AppliChem membranes can be UV Crosslinked by following the manufacturer's instructions. It is particularly recommended when working with short fragments, small samples, or low numbers of base pairs, because of the improved resolution this technique offers. Protein Blotting Pure Nitrocellulose Unsupported and PVDF-Star membranes are recommended for use in protein blotting. Nitrocellulose membranes are able to be more thoroughly blocked, reducing the high background potential associated with protein blotting. PVDF membranes are more resistant to the harsh chemicals used in Edman degradation. Alkaline Blotting For more rapid transfers, an alkaline blotting procedure can be used with Reprobe Nylon membranes. Alkaline blotting is not recommended when reprobing is required. See Protocol 6 for more details on Alkaline Blotting with Reprobe Nylon Positively Charged Transfer Membrane. Staining Procedures Reprobe Nitrocellulose Supported, Pure Nitrocellulose Unsupported and PVDF-Star membranes are recommended for procedures that require a staining step with India Ink, Coomassie Blue, Colloidal Gold, or any other commonly used stain. Nylon membranes irreversibly bind many stains. Reducing Backgrounds There are many sources of background problems, or low signal-to-noise ratios. Some of the most common include: contaminated probes, contaminated hybridization solutions, and incorrectly chosen stringency levels. Nonfat milk should not be used as a blocking agent as it may increase nonspecific binding. Nylon membranes have a much higher binding capacity than nitrocellulose membranes, and a greater potential for backgrounds. AppliChem membranes are all manufactured by strict quality control procedures, ensuring a uniform membrane with consistently low backgrounds. Troubleshooting Common Blotting Problems Many blotting problems can be eliminated by observing the following recommendations. Blotchy or incomplete transfers are caused by poor contact between the gel and the membrane. Even after careful smoothing of the membrane to the gel, incomplete degassing of transfer solutions can cause air pockets to form. Evolving gas from Tris or, in the case of protein transfers, methanol, can disrupt the tight contact necessary between the membrane and the gel for successful transfers. Smeared or skewed bands are often caused by uneven contact between the gel and the membrane, or the membrane and the chromatography paper. To avoid this problem, roll a pipet down the membrane after it has been applied to the gel, and once again over the chromatography paper after it has been applied to the membrane. Do not move the membrane until the transfer is complete, as this will cause smearing. © 2008 AppliChem • Transfer Membranes
Page 1 and 2: Transfer Membranes Take the Pink Li
Page 3 and 4: c o n t e n t s Introduction 2 Repr
Page 5 and 6: Reprobe Nitrocellulose Supported Tr
Page 7 and 8: Reprobe Nylon Positively Charged Tr
Page 9: Transfer Membranes Supplied by Appl
Page 13 and 14: Background Problems Reprobe Nylon P
Page 15 and 16: Protocols and Technical Papers - Su
Page 17 and 18: Protocols for DNA and RNA Applicati
Page 19 and 20: High Temperature Hybridization Solu
Page 21 and 22: Prehybridization This step should a
Page 23 and 24: Protocol 3: Southern (DNA) and Nort
Page 25 and 26: Clean probe solutions by adding a s
Page 27 and 28: Hybridization Convert the probe to
Page 29 and 30: Washes When the hybridization is co
Page 31 and 32: Protocol 7: Colony Hybridizations a
Page 33 and 34: Protocol 9: Colony Hybridization an
Page 35 and 36: Protocol 11: Oligonucleotide Probes
Page 37 and 38: Protocol 13: Nucleic Acid Dot/Slot
Page 39 and 40: Protocol 15: Western Blotting from
Page 41 and 42: Protocol 16: Protein Sequencing Fro
Page 43 and 44: Colloidal Silver Enhancement Use 77
Page 45 and 46: Protocol 22: Blocking with Blocking
Page 47 and 48: Prod. No. Description Comments A139
Page 49 and 50: Denhardt's - Solution (50X) Composi
Page 51 and 52: Salmon sperm DNA sodium salt Deoxyr
Page 53 and 54: Pure Nylon Neutral Transfer Membran
Page 56: A25 4t Matthes + Traut · Darmstadt

Transfer Membranes

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?