Latest revision as of 19:18, 17 October 2014

Team SUSTC-Shenzhen

Notebook

Element of an endeavor

A-B Toxin

2014 --to purify the protein

Protocol

Supplies and reagents

Acrylamide solutions
- 12% resolving gel(because our protein separately are 43kDa and 69kDa, so we should use 12% resolving gel)

components\volume	5ml	10ml	15ml	20ml	25ml	30ml	40ml	50ml
H2O	1.6	3.3	4.9	6.6	8.2	9.9	31.2	16.5
30%acrylamide mix	2.0	4.0	6.0	8.0	10.0	12.0	16.0	20.0
Tris-Cl(1.5M,PH8.8)	1.3	2.5	3.8	5.0	6.3	7.5	10.0	12.5
SDS(10%)	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
10%ammonium persulfate	0.05	0.1	0.15	0.2	0.25	0.3	0.4	0.5
TEMED	0.002	0.004	0.006	0.008	0.01	0.012	0.016	0.02

- 5% stacking gel

Components\Volume	1ml	2ml	3ml	4ml	5ml	6ml	8ml	10ml
H2O	0.68	1.4	2.1	2.7	3.4	4.1	5.5	6.8
30%acrylamide mix	0.17	0.33	0.5	0.67	0.83	1.0	1.3	1.7
Tris-Cl(0.5M,PH6.8)	0.13	0.25	0.38	0.5	0.63	0.75	1.0	1.25
SDS(10%)	0.01	0.02	0.03	0.04	0.05	0.06	0.08	0.1
10%ammonium persulfate	0.01	0.02	0.03	0.04	0.05	0.06	0.08	0.1
TEMED	0.001	0.002	0.003	0.004	0.005	0.006	0.008	0.01

Coomassie Brilliant Blue
- Coomassie Brilliant Blue R-250 1.25g, alcohol 250ml, acetic acid 80ml and ddH2O 670ml.
destain buffer
- 40%alcohol+10%acetic acid
ammonium persulfate in 10ml of H2O(10%w/v) and store at 4ºC
10%SDS
®Invitrogen Protein standard molecular-weight marker
®Invitrogen 5X SDS gel loading buffer
TEMED(electrophoreis grade)
Tris-HCl(0.5M,PH6.8),250ml
Tris-HCl(1.5M,PH8.8),250ml

Pouring gel

assembly the glass plate and fixation the equipment
pour the resolving gel, and keep a suitable distance from the opening.
cover a shell of ddH2O on the resolving gel until it solidify
remove the water shell and add the stacking gel.
add a Teflon comb into the stacking gel, when it become solid, remove it

Preparation of sample

prepare the samples in the appropriate volume of SDS gel-loading buffer and heat them into 100ºC for 3mins to denature the proteins

Running the SDS-PAGE gel

add a suitable volume of denatured samples into the hole of gel.
run the gel machine.
when the process finished, remove the glass plate
use the Coomassie Brilliant Blue to stain the gel
use the destaining solution to wash for about 1hrs
use the ddH2O to heat in 100ºC for 10mins.

@@ Line 12: / Line 12: @@
         date=2014|
         goal=--to purify the protein}}
-=protocol=
+=Protocol=
-==supplies and reagents==
+==Supplies and reagents==
-#Acrylamide solutions
+*Acrylamide solutions
-#*12% resolving gel(because our protein separately are 43kDa and 69kDa, so we should use 12% resolving gel)
+**12% resolving gel(because our protein separately are 43kDa and 69kDa, so we should use 12% resolving gel)
 <html><div class="table-responsive"></html>
@@ Line 36: / Line 36: @@
 <html></div></html>
-#*5% stacking gel
+**5% stacking gel
 <html><div class="table-responsive"></html>
@@ Line 56: / Line 56: @@
 |}
 <html></div></html>
-#Coomassie Brilliant Blue
+*Coomassie Brilliant Blue
-#*Coomassie Brilliant Blue R-250 1.25g, alcohol 250ml, acetic acid 80ml and ddH2O 670ml.
+**Coomassie Brilliant Blue R-250 1.25g, alcohol 250ml, acetic acid 80ml and ddH2O 670ml.
-#destain buffer
+*destain buffer
-#*40%alcohol+10%acetic acid
+**40%alcohol+10%acetic acid
-#ammonium persulfate in 10ml of H2O(10%w/v) and store at 4ºC
+*ammonium persulfate in 10ml of H2O(10%w/v) and store at 4ºC
-#10%SDS
+*10%SDS
-#®Invitrogen Protein standard molecular-weight marker
+*®Invitrogen Protein standard molecular-weight marker
-#®Invitrogen 5X SDS gel loading buffer
+*®Invitrogen 5X SDS gel loading buffer
-#TEMED(electrophoreis grade)
+*TEMED(electrophoreis grade)
-#Tris-HCl(0.5M,PH6.8),250ml
+*Tris-HCl(0.5M,PH6.8),250ml
-#Tris-HCl(1.5M,PH8.8),250ml
+*Tris-HCl(1.5M,PH8.8),250ml
-==pouring gel==
+==Pouring gel==
-#assembly the glass plate and fixation the equipment
+*assembly the glass plate and fixation the equipment
-#pour the resolving gel, and keep a suitable distance from the opening.
+*pour the resolving gel, and keep a suitable distance from the opening.
-#cover a shell of ddH2O on the resolving gel until it solidify
+*cover a shell of ddH2O on the resolving gel until it solidify
-#remove the water shell and add the stacking gel.
+*remove the water shell and add the stacking gel.
-#add a Teflon comb into the stacking gel, when it become solid, remove it
+*add a Teflon comb into the stacking gel, when it become solid, remove it
-==preparation of sample==
+==Preparation of sample==
 prepare the samples in the appropriate volume of SDS gel-loading buffer and heat them into 100ºC for 3mins to denature the proteins
-==running the SDS-PAGE gel==
+==Running the SDS-PAGE gel==
-#add a suitable volume of denatured samples into the hole of gel.
+*add a suitable volume of denatured samples into the hole of gel.
-#run the gel machine.
+*run the gel machine.
-#when the process finished, remove the glass plate
+*when the process finished, remove the glass plate
-#use the Coomassie Brilliant Blue to stain the gel
+*use the Coomassie Brilliant Blue to stain the gel
-#use the destaining solution to wash for about 1hrs
+*use the destaining solution to wash for about 1hrs
-#use the ddH2O to heat in 100ºC for 10mins.
+*use the ddH2O to heat in 100ºC for 10mins.
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