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Protocol for Western BlottingProtocol for Western Blotting
RED TEXT indicates important optimized settings
SDSPAGE separation
1 Make appropriate percentage of separation gel according to the MW of target proteins Related
recommendations and routine recipes of separationstacking gels are presented in the end
Tip 1 TrisTricine gels separate low MW proteins(less than 20 kDa) better than TrisGlycine gels
Tip 2 Gradient polyacrylamide gels can provide sharper bands and they separate a broader range of MW sizes on one gel
such as 10500 kDa
2 Prepare 30 μg C6 lysate with RIPA buffer by mixing 4X SDS sample buffer with lysate sample according to
the protein concentration measured by Bradford or BCA protein assay
3 Heat at 95100°C for 5 min Set up the electrophoresis apparatus with SDSpage gels or Tricine gels
4 Load samples and protein markers onto the gel Set 80 V to run the stacking gel increase to 120 V for the
separation gel till the end
Tip 3 Load enough sample for the first trial and adjust experiment system after getting target signal
Electrotransfer
5 In general PVDF membranes (or PSQ membrane with 022 μm micropore when MW target is less than 30
kDa) are recommended Soak membranes in methanol for 30 sec and then in the transfer buffer Soak the
filter papers and sponges in the transfer buffer as well
6 Sequentially assemble the transfer sandwich according to the illustration and make sure no bubbles are
trapped Apply semidry or wet transfer systems according to manufacturer’s instructions
Tip 4 If target MW is larger than 100 kDa wet transfer at 4 °C overnight is suggested instead of semidry method
Additional 01 SDS in the wet transfer buffer is recommended to facilitate transferring
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Immunoblotting
7 After transferring wash the membrane twice with distilled water If desired stain the membrane with
commercial Ponceau red solution for 1 min to visualize the protein bands mark the MW markers on the
membrane with a pencil then wash up the staining with 1X TBST
8 Block with 1X TBST containing 5 nonfat dry milk (or 15 BSA for phosphoepitope antibodies) with
agitation at room temperature for 1 h
9 Dilute primary antibody (250011AP AUTS2 Rabbit PolyAb) with a starting dilution ratio of 1600 in
blocking solution Optimal dilution should be determined in pretests Incubate membrane with the primary
antibody with agitation at room temperature for 15 hours Wash membrane 3 times with 1X TBST for 10
min each
10 Incubate the membrane with the HRPconjugated secondary antibody diluted at 15000 in blocking
solution at room temperature for 1 h Wash membrane 3 times with 1X TBST for 10 min each
Tip 5 Do not let the membrane dry for the entire process 02 NaN3 could be included in the primary antibody dilution
for preservation but never in the secondary antibody dilution
Signal Detection
11 Prepare ECL substrate according to the manufacturer’s instructions
12 Incubate the membrane completely with substrate for 15 min
13 Expose autoradiography film in a dark room or exposure membrane under a chemiluminescence imaging
system Perform multiple exposures to determine the optimal one (60180 Sec exposure to get the fig)
14 Remember to mark the protein MW markers on the film
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ELISAEnzymelinked immunosorbent assay WBWestern Blotting IHCImmunohistochemistry ICCImmunocytochemistry
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Solutions
4X SDS sample buffer 50 ml 1X TBST 1000 ml
250 mM Tris•HCl (pH 70) (1M stock) 125 ml 20 mM Trisbase 24 g
40 glycerol 20 ml 150 mM NaCl 87 g
5 SDS 25 g 02 Tween20 2 ml
002 Bromophenol Blue 10 mg Adjust pH to 76
5 βmercaptoethanol 25 ml Add ddH2O to 1000ml
Add ddH2O to 50ml aliquot and store at 20°C
1X Running buffer 1000 ml Semidry transfer buffer 1000 ml
125 mM Trisbase 151 g 48 mM Trisbase 581 g
100 mM Glycine 75 g 39 mM Glycine 293 g
005 SDS 05 g 00375 SDS 0375 g
Add ddH2O to 1000 ml 20 Methanol 200 ml
Add ddH2O to 1000 ml
Wet transfer buffer 1000 ml Tricine gel running buffer 1000 ml
25 mM Trisbase 303 g 100 mM Trisbase 121 g
192 mM Glycine 144 g 100 mM Tricine 179 g
20 Methanol 200 ml 01 SDS 1 g
Add ddH2O to 1000 ml Add ddH2O to 1000ml
Related Products in PROTEINTECH
Product Name Catalog No Size Application
HRPconjugated AffiniPure Goat AntiMouse Ig(G+L) SA000011 100 μl ELISA WB IHCICC
HRPconjugated AffiniPure Goat AntiRabbit Ig(G+L) SA000012 100 μl ELISA WB IHCICC
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Recipes for SDSPAGE GelRecipes for SDSPAGE Gel
A For MW of target protein between 20200 kDa conventional SDSPAGE gel could be configured
following recipes in the table below
ITEMSITEMS Separation Gel (10ml)Separation Gel (10ml) Stacking Gel (ml)Stacking Gel (ml)
MW of target proteinMW of target protein 80200 kDa 35100 kDa 2560 kDa 2040 kDa 4ml 6ml 8ml
Gel percentageGel percentage 8 10 12 14 4 4 4
ddHddH22OO 46 40 33 23 29 43 57
30 Acrylamide30 Acrylamide 27 33 4 5 05 08 11
15 M Tris•HCl (pH88)15 M Tris•HCl (pH88) 25 25 25 25
10 M Tris·HCl (pH68)10 M Tris·HCl (pH68) 05 08 1
10 SDS10 SDS 01 01 01 01 004 006 008
10 APS10 APS 01 01 01 01 004 006 008
TEMEDTEMED 001 001 001 001 0004 0006 0008
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B For MW of target protein less than 20 kDa tricine gel system is greatly suggested to obtain
higher resolution Make three layers of tricine gels as in the following table apply specific tricine
gel running buffer to the running system and perform transfer as usual
ITEMsITEMs SeparationSeparation IntermediateIntermediate StackingStacking
Gel percentageGel percentage 15 10 4
Gel volumeGel volume 6 ml 3 ml 2ml
38 Glycerol solution38 Glycerol solution 16 —
ddHddH22OO — 12 14
30 Acrylamide30 Acrylamide 27 08 03
30 M Tris·HCl (pH85)30 M Tris·HCl (pH85) 214 1
10 M Tris•HCl (pH68)10 M Tris•HCl (pH68) — — 03
10 SDS10 SDS 006 003 002
10 APS10 APS 006 003 002
TEMEDTEMED 0003 0003 0002
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