Ketner: Tissue culture viral lysate Western Blot - Revision history

Crp724: /* Materials */

Crp724 — Wed, 06 Sep 2006 18:03:20 GMT

Materials

Crp724 — Wed, 06 Sep 2006 18:00:05 GMT

Procedure

←Older revision		Revision as of 18:00, 6 September 2006
Line 8:		Line 8:
	*[[PBS-T]]		*[[PBS-T]]
	*5% milk in PBS-T		*5% milk in PBS-T
		+
		+
		+	----

	===Procedure===		===Procedure===
Line 39:		Line 42:
	*Wash 3x with 5% milk in PBS-T for 5 min each		*Wash 3x with 5% milk in PBS-T for 5 min each
	*Wash 1x with PBS-T for 5 min		*Wash 1x with PBS-T for 5 min
		+
		+	====Develop Blot and document====
		+	specific to secondary antibody

Crp724 — Wed, 06 Sep 2006 17:59:03 GMT

Probe Blow with antibodies

←Older revision		Revision as of 17:59, 6 September 2006
Line 32:		Line 32:
	*overnight, constant voltage of 40mA		*overnight, constant voltage of 40mA

-	====Probe ~~Blow~~ with antibodies====	+	====Probe Blot with antibodies====
	*Block with 5% milk in PBS-T for 1hr		*Block with 5% milk in PBS-T for 1hr
	*Primary in 5% milk in PBS-T for 2 hrs		*Primary in 5% milk in PBS-T for 2 hrs

Crp724 — Wed, 06 Sep 2006 17:58:50 GMT

Procedure

←Older revision		Revision as of 17:58, 6 September 2006
Line 30:		Line 30:
	====Transfer to membrane====		====Transfer to membrane====
	specific for apparatus		specific for apparatus
		+	*overnight, constant voltage of 40mA
		+
		+	====Probe Blow with antibodies====
		+	*Block with 5% milk in PBS-T for 1hr
		+	*Primary in 5% milk in PBS-T for 2 hrs
		+	*Wash 3x with 5% milk in PBS-T for 5 min each
		+	*Secondary in 3% milk in PBS-T for 45 min
		+	*Wash 3x with 5% milk in PBS-T for 5 min each
		+	*Wash 1x with PBS-T for 5 min

Crp724 — Wed, 06 Sep 2006 17:55:45 GMT

Transfer to membrane

Crp724 — Wed, 06 Sep 2006 17:46:31 GMT

=Transfer to membrane

Crp724 — Wed, 06 Sep 2006 17:46:21 GMT

Procedure

Crp724 — Wed, 06 Sep 2006 17:45:58 GMT

Prepare Gel

←Older revision		Revision as of 17:45, 6 September 2006
Line 24:		Line 24:
	*Layer isopropanol on top to remove bubbles		*Layer isopropanol on top to remove bubbles
	*[[When hardened create stacking gel]]		*[[When hardened create stacking gel]]
		+	*Cast stacking gel with comb, remove comb when hardened
		+	*Fill with 1x running buffer
		+	*Load samples and run at 50-120V

Crp724 — Wed, 06 Sep 2006 17:43:19 GMT

Prepare Gel

←Older revision		Revision as of 17:43, 6 September 2006
Line 21:		Line 21:
	*Boil agar to create plug		*Boil agar to create plug
	*Drip agar down side of cassette with pasteur pipette, just enough to form seal		*Drip agar down side of cassette with pasteur pipette, just enough to form seal
-	*[[Create running]]	+	*[[Create running gel]]
	*Layer isopropanol on top to remove bubbles		*Layer isopropanol on top to remove bubbles
	*[[When hardened create stacking gel]]		*[[When hardened create stacking gel]]

Crp724 — Wed, 06 Sep 2006 17:43:04 GMT

Prepare Gel

←Older revision		Revision as of 17:43, 6 September 2006
Line 21:		Line 21:
	*Boil agar to create plug		*Boil agar to create plug
	*Drip agar down side of cassette with pasteur pipette, just enough to form seal		*Drip agar down side of cassette with pasteur pipette, just enough to form seal
-	*[[Create running ~~gel and pour to 2/3 height~~]]	+	*[[Create running]]
	*Layer isopropanol on top to remove bubbles		*Layer isopropanol on top to remove bubbles
	*[[When hardened create stacking gel]]		*[[When hardened create stacking gel]]