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Electroblotting Procedure

Electroblotting Procedure
 
Electrophoresis
The electrophoresis apparatus can be of any manufacturer. The gel apparatus used in the BSI is manufactured by Bio Rad. The electrophoresis is carried out using the method of Laemmli(Nature:277,680,1970). Several other gel systemshave been used with no problem.
 
Electroblotting
The electroblotting apparatus that have been used in the facility are all Bio Rad tank and semidry units.
 
Electroblotting Membranes
Membranes from many manufacturers have been evaluated by transfer of known proteins in quantitated samples. The following membranes have given satisfactory N-terminal sequencing and internal sequencing results.
 
Product Source Part#
Problott Applied Biosystems Inc. 400994
Immobilon PSQ Millipore Corp. ISEQ20200
PVDF Bio Rad 162-0182
 
Chemicals
Product Source Part#
Methanol Burdick and Jackson 230-4
Acetonitrile Burdick and Jackson 015-4
Trifluoroacetic Acid Pierce Chemical Co, 28903
CAPS (3-[cyclohexylamino]-1-propane-sulfonic acid Sigma C-2632
Ponceau S Sigma P-7767
Coomassie R-250 Sigma B-0630
Bromophenol blue Bio Rad 161-0404
Acetone J.T. Baker 9017-02
Tween 20 Aldrich 27,434-8
Ammonium persulfate Bio Rad 161-0700
Glycine Bio Rad 161-0717
N,N’-methylene bis-acrylamide Bio Rad 161-0200
N,N,N,N,-tetra-methyleneamine Bio Rad 161-0800
Acrylamide Bio Rad 161-0100
Sodium Bicarbonate J.T. Baker 3003-01
2-mercaptoethanol Sigma M-6250
N-ethylmorpholine Pierce 20805
Glycerol Fisher BP229-1
Acetic Acid J.T. Baker 9508-04
Amido Black Sigma A-8181
 
Electroblotting Method
The transfer is performed by the method of Matsudaira(J.Biol.Chem.;262,10035,1987).
CAPS working solution:
  Stock Solution 10X
  22.13g of CAPS in D.I. water 800 mL
  pH to 11.0 with NaOH
  adjust volume to 1000 mL
     
Working Solution 1X
  200 mL of Stock 10X CAPS added to 200 mL MeOH and 1600 mL D.I. water
     
Cut PVDF membrane to the size of the gel
Wet membrane with methanol for a few seconds.
Soak membrane in blotting buffer for five minutes
Soak gel for five minutes in blotting buffer
Soak pads and filters in blotting buffer and assemble the transblott unit
Electroblott at 50 volts constant voltage for 20 minutes to one hour (for proteins from 80-100,000 and larger delete the methanol)
Remove the membrane and the gel from the sandwich rinse with D.I. water
 
Staining
All conventional methods of staining may be employed with PVDF membranes with slight modifications as below.
Coomassie R-250:
  Soak blott(after D.I. water rinse) in methanol
  Stain PVDF membrane with 0.1% Coomassie R-250 in 40% MeOH for no longer than ONE MINUTE usually 15 to 20 seconds will suffice. (Staining for longer periods of time will result in high background and will interfere with extraction and cleavage)
  Destain with 50% methanol, several changes
  Rinse extensively with D.I. water
  Cut out the band of interest
     
Ponceau S:
  Stain the PVDF membrane in 0.25% Ponceau S in 1% Acetic acid for one to three minutes(until protein bands are visible)
  Destain in D.I. water
  Excise the band of interest
  Rinse with D.I. water
     
Amido Black:
  Soak blott after D.I. water rinse with methanol
  Stain PVDF membrane with O.2% Amido Black in 40% MeOH for 30 seconds to one minute
  Destain in D.I. water with multiple changes until bands are clear and low background
  Cut out band of interest