transfer
in transfer buffer overnight 4'C 200mA
1.blocking in blocking buffer 1hour RT or overnight
2.TBS-T wash, 5min 3'
3.1st antibody 4'C overnight, 1st antibody is reused!
4.wash with blocking buffer 4min 4'
5.2nd antibody 40min RT
6.wash with TBS-T 4min 4'
7 wast with 1x PBS 5min
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TBS-T
1M Tris-Cl pH 8.0 10ml
5M NaCl 30ml
tween 20 20% 2.5ml
total 1L
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blocking buffer
1M Tris-Cl pH 8.0 25ml
5M NaCl 25ml
tween 20 20% 2.5ml
dry milk 40g
total 1L
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stripping buffer 50ml
2-Mercaptoehanol 350ul
20% SDS 5ml
1M Tris-HCl pH6.7 3.12ml
1. 40 min at 50C
2.TBST wash 10min 3'
3.blocking........
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Transfer Buffer 4L
Tris 12g
glycine 57.6g
Methanol 800ml
pH should between 8.2~8.4
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SDS-PAGE
12% 10 ml
Water 3.3ml
30% acrylamide 4.0ml
1.5M Tris 2.5ml
10% SDS 100ul
10% APS 100ul
TEMED 4ul
16% 10ml
Water 2.0ml
30% acrylamide 5.3ml
1.5M Tris 2.5ml
10% SDS 100ul
10% APS 100ul
TEMED 4ul
stack 5ml
Water 3.4ml
30% acrylamide 550ul
60% giyceral 410ul
4X upper buffer 650ul
10% APS 37.5ul
TEMED 5ul
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前實驗室的配方
現在實驗室直接買做好的PAGE