Bio-rad Trans-Blot® Cell User Manual

Trans-Blot®ElectrophoreticTransfer CellInstructionManualCatalog Numbers170-3910, 170-3925,170-3926, 170-3939,170-3945, 170-3946,170-3950, 170-3951170-

2.2 High Intensity Field OptionThe electrode cards of the Trans-Blot cell can be moved closer together to allow higherfield strengths (V/cm), and thus

2.3 Acidic TransfersIf transferring under acidic conditions, switch the gel and membrane in the set up instruc-tions. This will place the membrane on

Table 3.2. DNA and RNAStandard Field Standard Field High Intensity FieldOvernight 8 cm electrode distance 4 cm electrode distance❄COOLING REQUIREDTAE

2. Pre-equilibration of gelsAll electrophoresis gels should be pre-equilibrated in transfer buffer prior to electrophoretictransfer. Pre-equilibration

8. Transfer buffer recommendationsUse only high quality, reagent grade methanol. Contaminated methanol can result inincreased transfer buffer conducti

126. 1.0x TBE (Tris-Borate EDTA) pH 8.390 mM Tris-Borate 1 mM EDTA5x stock solution54 g Tris base27.5 boric acid20 ml 0.5 m EDTA (pH 8.0)Add 200 ml 5x

5. Vary buffer type and pHa. Maximize charge-to-mass ratio. It appears that alcohols present in SDS transfer bufferstrip SDS from proteins. Basic prot

Section 5Choice of Blotting Membranes5.1 Protein Blotting MembranesNitrocellulose MembraneNitrocellulose membranes have been used extensively for prot

Table 5.1 Guide to Protein Blotting MembranesA variety of blotting membranes is available for immunoblotting, each with particular advan-tages dependi

6. Power supply circuit is inoperative, or an inappropriate power supply wasused.• Check the fuse. Be sure the voltage and current output of the power

Note1. Assembly & DisassemblyTo insure best performance from the Trans-Blot electrophoretic transfer cell, becomefully acquainted with these opera

4. The gel electrophoresis may be at fault.• Artifacts of electrophoresis may be produced by poor polymerization,inappropriate running conditions, con

Immune-Specific DetectionOverall High Background1. Blocking conditions are inappropriate.• Match the blocker to the membrane. For example, nylon and P

No Reaction or Weak Signal1. The sample load was insufficient.• Increase the amount of protein applied. Concentration of the sampleprior to loading ma

3. Activity test for the first antibody solution.• Use an ELISA, RID, Ouchterlony immunodiffusion, or precipitation testto determine reactivity of the

Biotin-Blot Total Protein Detection — High Background1. Blocking conditions are insufficient.• Match the blocker to the membrane. Nylon membranes requ

Section 7Product InformationCatalogNumber Product Description170-3939 Trans-Blot Cell with Plate Electrodes, complete, includes 2 GelHolder cassettes,

9. Stellwag, E. J. and Dahlberg, A. E., Nuc. Acids Res., 8, 299 (1980).10. Kutateladze, T. V., Axelrod, B. D., Gorbulev, V. G., Belzhelarshaya, S. N.

Bio-RadLaboratoriesAustralia,Bio-Rad Laboratories Pty Limited, Block Y Unit 1, Regents Park Industrial Estate, 391 Park Road, Regents Park, NSW 2143•P

Table of ContentsSection 1 Introduction...11.1 Specific

Section 1IntroductionBlotting was first performed by Southern1in 1975 with the transfer of DNA from agarosegels to nitrocellulose membranes. Blotting

1.2 Safety InstructionsPower to the Trans-Blot cell is supplied by an external DC voltage power supply. Thispower supply must be ground isolated in su

Trans-Blot Cell Description & Assembly of Parts3LidFiber padFilter paperMembraneGelFilter paperFiber padGel holdercassetteSupercooling coilPlate c

Section 2Trans-Blot Cell Assembly2.1 Preparation for Blotting1. Prepare the transfer buffer. (See Section 3.3 for buffer formulation. Using buffer chi

54. Close the cassette firmly, being careful not to move the gel and filter paper sandwich.Lock the cassette closed with the white latch.5. Place the

7. Addition of a standard stir bar will help to maintain even buffer temperature and ion dis-tribution in the tank. Set the speed as fast as possible