REQ: EMSA (gelshift) protocol

Heinz-Juergen Schaefers mfm434 at rzmail.uni-erlangen.de
Tue Jul 16 15:27:08 EST 1996


Hi all!

As requested by several netters, I post the complete EMSA protocol:

Electrophoretic Mobility Shift Assay (EMSA, Gelshift)

1.) solutions and buffers:

	solution A:		10 mM Hepes, pH 7.9
				10 mM KCl
				0.1 mM EDTA
				0.1 mM EGTA
			store at 4°C
	solution A*:		add right before use 1 µL 1M DTT and 5 µL 100mM PMSF
				 per mL solution A

	solution C:		20 mM Hepes, pH 7.9
				25 % Glycerol
				0.4 M NaCl
				1 mM EDTA
				1 mM EGTA
			STORE AT 4°C
	solution C*:		add right before use 1 µL 1M DTT and 5 µL 100mM PMSF
				 per mL solution C

	binding buffer:		120 mM Hepes, pH 7.9
				60 mM KCl
				30 mM MgCl2
				40 mM Tris, pH 8.0
				6 mM EDTA, pH 8.0
				ad H2Odd to 2 mL final volume

	TETG 1N:		50 mM Tris, pH 7.5
				10 mM EDTA, pH 7.5
				100 mM NaCl
				0.1 % Tween
				15 % Glycerol
				ad H2Odd to 2 mL final volume

	poly d(I-C):	dilute to 2 µg/mL in H2Odd
		(Boehringer Mannheim: 10 A260 units = 350 µg)

	inkubation buffer:	160 µL binding buffer
				640 µL TETG 1N
				80 µL poly d(I-C)
				80 µL ß-mercapto ethanol
				400 µL H2Odd


2.) collecting cells:

	all steps on ice, use cold solutions !!!
	4-6 petri dishes (10 cm diameter) of nearly confluent cells (20-30*10(6) cells) per point
	-   wash cells with cold PBS, collect the cells in PBS with a rubber policeman,
	     pool all cells of one point
	-   zentrifuge 10 min 1200 rpm 4°C, discard supernatant
	-   resuspend cells in 1 mL PBS, transfer cell suspension to eppendorf cap
	-   zentrifuge 15 sec 13000 rpm, discard supernatant
	-   add 300 µL solution A*, resuspend with yellow pipet tip
	-   15 min on ice
	-   add 20 µL 10% Nonidet P-40 (NP-40)
	-   vortex 10 sec
	-   zentrifuge 1 min, discard supernatant
	-   add 30 µL ice-cold solution C*, resuspend
	-   vortex vigorously 20 min in a cold room
	-   zentrifuge 5 min 13000 rpm 4°C
	-   aliquot supernatant (10 µL aliquots)
	-   measure protein content:
		5 µL supernatant
		75 µL H2Odd
		20 µL BioRad dye
		(as standard: 0-10 µg/mL BSA in H2Odd)
		[use less supernatant and more H2Odd if necessary, both together 80 µL!!]
	-   freeze aliquots in liquid nitrogen
	-   store aliquots at -70°C


3.) labelling:

	4 ng oligo  (25 to 50 bp is the best length, TF site in the center of the oligo)
	2 µL 10x T4-kinase buffer
	1 µL 32P-gamma-ATP
	2 µL T4 polynucleotide kinase
	ad H2Odd to a final volume of 20 µL

	-   incubate 30 min at 37°C
	-   separate bound and free activity by using Sephadex G50 fine columns 
	    (Pharmacia or Boehringer)
	-   measure aliquot of the labelled_oligo_containing_flowthrough in a liquid
	    scintillation counter
	-   dilute to 50000 cps per µL with H2Odd


4.) binding:

	17 µL inkubation buffer
	1 µL labelled oligo (= 50000 cps)
	5 µg protein
	(x µL 50x unlabelled oligo (for competition) or antibody (for supershift))
	add H2Odd to a final volume of 25 µL

	-   incubate 30 min at 4°C or room temperature
	    (depends on the oligo/binding protein you want to examine, you have to try)
	-   ad 1 µL 0.1% BPB (bromine-phenol-blue)
	-   load onto a 6 % non-denaturating (without SDS!) PAA gel (~ 15*20 cm)
		running buffer: 1x TBE
	-   run gel 30 min at 22 mA, followed by 2 to 3 hrs at 25 mA

after run:
	-   place gel on a piece of Whatman paper, cover gel with saran wrap
	-   dry gel in a vacuum drier for 1.5 to 2 hrs at 80°C
	-   auto-radiograph dry gel at -70°C


bye
	Heinz
-- 
Dipl. Biol. Heinz-Juergen Schaefers
Universitaet Erlangen-Nuernberg / Med. Klinik IV / Nephrologisches Forschungslabor
Loschgestr. 8, D-91054 Erlangen, Germany
E-mail: dienstl.: mfm434 at rzmail.uni-erlangen.de / priv.: h-j.schaefers at erlangen.netsurf.de



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