Difference between revisions of "Anti-BrDU Antibody labeling"

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(Material)
(Material)
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== Material ==
 
== Material ==
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Centricon-100
 
Centricon-100
  
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1) Concentrate the antibody to approximately 1.5 mg/ml and dialyze the antibody into 0.1 M Borate buffer pH 8.8 if necessary
 
  
2) Warm the Rhodamine NHS (Rh NHS) to room temp and then dissolve in DMSO at 0.5 mg/ml
+
== Procedure ==
 +
 
 +
# Concentrate the antibody to approximately 1.5 mg/ml and dialyze the antibody into 0.1 M Borate buffer pH 8.8 if necessary
 +
 
 +
# Warm the Rhodamine NHS (Rh NHS) to room temp and then dissolve in DMSO at 0.5 mg/ml
  
3) Spin the Rh NHS solution in the microfuge at 13,000 rpm for 2 minutes to remove insoluble material.
+
# Spin the Rh NHS solution in the microfuge at 13,000 rpm for 2 minutes to remove insoluble material.
  
4) Add the Rh NHS solution to the antibody at a ratio of  1:20--1:60 (vol Rh NHS: vol AB).  This means 17-50 µl of Rh NHS per 1ml of antibody
+
# Add the Rh NHS solution to the antibody at a ratio of  1:20--1:60 (vol Rh NHS: vol AB).  This means 17-50 µl of Rh NHS per 1ml of antibody
 
Vol of antibody:
 
Vol of antibody:
 
Vol of Rh NHS:
 
Vol of Rh NHS:
  
5) Mix gently by inverting 2-3 times and  
+
# Mix gently by inverting 2-3 times and  
  
6) Incubate in the dark for 2-4 hours
+
# Incubate in the dark for 2-4 hours
  
7) Add 10 µl of 0.1M glycine per 1 ml of antibody  to quench the reaction
+
# Add 10 µl of 0.1M glycine per 1 ml of antibody  to quench the reaction
  
8) Incubate for 10 minutes
+
# Incubate for 10 minutes
  
9) Desalt into Borate buffer (TBS is also ok) using gel filtration columns (NAP5 or NAP10).   
+
# Desalt into Borate buffer (TBS is also ok) using gel filtration columns (NAP5 or NAP10).   
  
10) To store the antibody, add an equal volume of glycerol and store at -20 C.
+
# To store the antibody, add an equal volume of glycerol and store at -20 C.
  
  

Revision as of 08:16, 29 June 2015

Material

Centricon-100

Rhodamine N-hydroxy-succiniymidyl ester (stored at -20)

DMSO

Antibody in 0.1M Borate buffer pH 8.8

NAP desalting columns or dialysis membrane.

0.1M Borate buffer pH 8.8

0.1M glycine, pH 7


Procedure

  1. Concentrate the antibody to approximately 1.5 mg/ml and dialyze the antibody into 0.1 M Borate buffer pH 8.8 if necessary
  1. Warm the Rhodamine NHS (Rh NHS) to room temp and then dissolve in DMSO at 0.5 mg/ml
  1. Spin the Rh NHS solution in the microfuge at 13,000 rpm for 2 minutes to remove insoluble material.
  1. Add the Rh NHS solution to the antibody at a ratio of 1:20--1:60 (vol Rh NHS: vol AB). This means 17-50 µl of Rh NHS per 1ml of antibody

Vol of antibody: Vol of Rh NHS:

  1. Mix gently by inverting 2-3 times and
  1. Incubate in the dark for 2-4 hours
  1. Add 10 µl of 0.1M glycine per 1 ml of antibody to quench the reaction
  1. Incubate for 10 minutes
  1. Desalt into Borate buffer (TBS is also ok) using gel filtration columns (NAP5 or NAP10).
  1. To store the antibody, add an equal volume of glycerol and store at -20 C.


Notes: The Rhodamine N-hydroxy-succiniymidyl ester is highly reactive with water so if it gets wet before the experiment, the coupling reaction will not work well. To treat the Rhodamine N-hydroxy-succiniymidyl ester properly: Store with dessicant at -20 C. Before use, bring out the bottle to room temperature and allow it to warm up before opening the bottle to prevent condensation entering the bottle. Weigh out the powder and dissolve in fresh DMSO just before the reaction.