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2021-02-01T19:52:47Z

References

← Older revision		Revision as of 19:52, 1 February 2021
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	For 1ml of antibody solution add:		For 1ml of antibody solution add:

	[[Image:SAS_Calc.gif]], where		[[Image:SAS_Calc.gif\|link=Special:FilePath/SAS_Calc.gif]], where

	SF = final saturation, Si = initial saturation.  Example, for 45% of a 1ml starting volume with no prior SAS addition:		SF = final saturation, Si = initial saturation.  Example, for 45% of a 1ml starting volume with no prior SAS addition:
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	== References ==		== References ==
			<references />
	== <references /> ==

Admin: Created page with "== Buffers Required: ==

Note: THe volume of buffers prepared is dependent on the starting volume of antibody-containing media.  Adjust volumes accordingly.

=====..."

2021-02-01T19:52:13Z

Created page with "== Buffers Required: == <pre>Note: THe volume of buffers prepared is dependent on the starting volume of antibody-containing media. Adjust volumes accordingly. </pre> =====..."

New page

== Buffers Required: ==
<pre>Note: THe volume of buffers prepared is dependent on the starting volume of antibody-containing media. Adjust volumes accordingly.
</pre>
===== PBS: =====

To 800ml ddH2O add:

*8 g NaCl
*0.2 g KCl
*1.44 g Na2HPO4 (sodium phosphate dibasic)
*0.24 g KH2PO4 (monopotassium phosphate)

pH to 7.4, and bring up to 1L with ddH2O. Autoclave to sterilize.

===== Saturated Ammonium Sulphate (SAS): =====

To 1000 ml of double-distilled water add:

*766.8 g g Ammonium sulphate<ref>http://cshprotocols.cshlp.org/content/2006/1/pdb.tab1</ref><ref>http://research.biology.arizona.edu/mosquito/willott/proj/LabPro/Prot/Proppt/Ammon.html</ref><ref>http://www.science.smith.edu/departments/Biochem/Biochem_353/Amsulfate.htm</ref>

Dissolve at 50C to 60C.  Stand overnight at room temperature, then adjust pH to  6.8-7.0 with 5 or 10 M NaOH. Measure the pH using small aliquots.

===== Digestion Buffer: =====

Per 100 ml of water add:

*355 mg (20mM) sodium phosphate dibasic
*292 mg (10mM) EDTA

pH to 7.0.  Immediately before use add 20mM cysteine-HCl

===== 10mM Tris-HCl =====

To 100ml of water add

*158 mg Tris-HCl

pH to 7.5.

== IgG Fractionation ==

Based on <ref>http://onlinelibrary.wiley.com/doi/10.1038/npg.els.0003761/full#a0003761-tbl-0002</ref>

#Dilute antibody 1:2 with PBS on ice.  E.G. 10ml of hybridoma media would be diluted with 20ml of PBS.
#With gentle stirring, on-ice, add SAS drop-wise to reach a final concentration of 45% ammonium hydroxide (see calculator below).  '''Warning: stir gently.  Avoid foaming, as this indicates protein denaturation.'''
#Once SAS addition is complete stir for 30min on-ice, then centrifuge at 1000g for 15 min at 4°C.
#Wash the precipitate with 45% SAS, then re-pellet at 1000g for 15 min at 4°C.
#Redissolve the precipitate in the same volume of PBS as the original antibody solution.
#Centrifuge at 5000g for 15 min at 4°C to remove any particulates.
#Transfer the supernatant to a clean tube and reprecipitate immunoglobulin using 40% SAS
#Centrifuge at 1000g for 15 min at 4°C.
#Redissolve the precipitate in a minimum volume of digestion buffer without cysteine (0.5 mL) and dialyse against 5L of digestion buffer without cysteine at 4°C overnight.
#Centrifuge at 5000g for 15 min at 4°C to remove more insoluble material.
#Spec the protein to determine the concentration.

===== SAS Volume Calculator: =====

For 1ml of antibody solution add:

[[Image:SAS_Calc.gif]], where

SF = final saturation, Si = initial saturation.  Example, for 45% of a 1ml starting volume with no prior SAS addition:

Volume (ml) = 1(0.45-0) / (1-0.45) = 0.82ml

 

== Fab Preparation ==

#Add 20mM cystein-HCl to the fractionated IgG & digest buffer.
#Dilute fractionated IgG to a concentration of 10mg/ml in digest buffer.
#Mix immobilized papain beads (Thermo #20341) to obtain an even suspension.  Take 500ul of slurry* for every 1ml of diluted IgG to be processed.  Transfer slurry to a clean tube large enough to perform the digest in.
#Wash gel 2X in 4ml/500ul of slurry with digest buffer.  Centrifuge briefly (1-2 min at 250 x g) to pellet papain.
#Pellet washed papain, and resuspend in 0.5ml of digest buffer per 500ul of starting slurry.
#Add antibody solution to papain slurry.
#For non-human IgG, incubate 5 hours to overnight at 37oC with mixing.  Four hours is sufficient for human IgG.
#For every 1ml of diluted antibody add 1.5ml 10mM tris-HCl solution, then centrifuge 1000g for 15 min at 4°C.
#Carefully draw supernatant off of papain pellet.

 

*Smaller amounts of papain may be used, but longer digests are required and incomplete digestion may occur as a result.  For antibody quantities less than 10mg, use 500ul of slurry.

 

== Fab Purification ==

Fab purification should be performed as per the instructions found in [[Fab FPLC|Fab purification using FPLC]].

 

== References ==

== <references /> ==

Fab preparation - Revision history

Admin: /* References */

Admin: Created page with "== Buffers Required: ==
Note: THe volume of buffers prepared is dependent on the starting volume of antibody-containing media. Adjust volumes accordingly.
=====..."