Western Blot Common Reagent Formulations-EnkiLife-Recombinant Proteins,Antibodies,Kits,Cell Biology

Common Western Blot Reagent Recipes

Lysis Buffers Buffer Preparation Electrophoresis Transfer Blocking Solutions

Common Lysis Buffer Recipes

Cytoskeleton Binding Protein Extraction Buffer

For extracting cytoskeleton-binding proteins

Tris, pH 7.4 10 mM

NaCl 100 mM

EDTA 1 mM

EGTA 1 mM

NaF 1 mM

Na₄P₂O₇ 20 mM

Na₃VO₄ 2 mM

Triton X-100 1%

Glycerol 10%

SDS 0.1%

Sodium deoxycholate 0.5%

Soluble Protein Buffer

For extracting soluble proteins

Tris-HCl, pH 7.5 20 mM

EGTA (Ca²⁺ chelator) 1 mM

RIPA Lysis Buffer

Suitable for nuclear membrane disruption to extract nuclear proteins

Tris HCl, pH 8 50 mM

NaCl 150 mM

NP-40 1%

Sodium deoxycholate 0.5%

SDS 0.1%

Contains the ionic detergent sodium deoxycholate, suitable for nuclear membrane disruption to extract nuclear proteins. May denature kinases and disrupt protein interactions.

NP-40 Lysis Buffer

Mild lysis buffer recipe

Tris HCl, pH 8 20 mM

NaCl 137 mM

Glycerol 10%

NP-40 1%

EDTA 2 mM

Common Reagent Preparation Methods

100 mM EDTA Stock Solution

EDTA solution preparation

EDTA 1.86g

Ultrapure water 40ml

NaOH Adjust pH to 7.4

Bring to volume 50ml

Storage condition 4℃

10×TBS Concentrate

TBS buffer concentrate

Trizma HCl 24.23 g

NaCl 80.06 g

pH adjustment 7.6 (HCl)

Bring to volume 1L ddH₂O

TBST Working Solution (1L)

Wash buffer

10×TBS 100 ml

Ultrapure water 900 ml

Tween20 1 ml

Phosphatase Inhibitor Stock Solution

Prevents protein dephosphorylation

Sodium orthovanadate 100mM

Solvent Double distilled water

pH adjustment 9.0 (HCl)

Treatment Boil until colorless

Storage condition -20℃

Perform in a fume hood, cover during boiling to prevent evaporation, discard if solution turns yellow. Common working concentration range is 1-10 mM.

Mild Stripping Buffer (1L)

Membrane regeneration buffer

Glycine 15 g

SDS 1 g

Tween20 10 ml

pH 2.2

Solvent Ultrapure water to 1L

Strong Stripping Buffer (100ml)

Strong membrane regeneration buffer

10% SDS 20 ml

0.5M Tris HCl pH 6.8 12.5 ml

Ultrapure water 67.5 ml

β-mercaptoethanol 0.8 ml

Operate in a fume hood

Antibody Dilution Buffer

For diluting primary or secondary antibodies

Normal serum 100 µl

BSA 10 mg

TBS 900 µl

Carbonate Coating Buffer (100mM, pH 9.6)

Coating buffer for ELISA experiments

Na₂CO₃ 3.03 g

NaHCO₃ 6.0 g

Solvent Ultrapure water to 1L

PBS (pH 7.4, 500ml)

Phosphate Buffered Saline

Na₂HPO₄ 1.16 g

KCl 0.1 g

KH₂PO₄ 0.1 g

NaCl 4 g

1 M Tris-HCl，pH 7.6

Tris buffer

Tris base 12.11 g

ddH2O 80 mL

pH adjustment 7.6 (HCl)

Bring to volume 100mL ddH2O

0.5 M Tris-HCl，pH 6.8

Tris buffer

Tris base 6.06 g

ddH2O 60 mL

pH adjustment 6.8 (HCl)

Bring to volume 100mL ddH2O

10% SDS

Sodium dodecyl sulfate solution

SDS 1g

ddH2O 10mL

1.0% Bromophenol Blue

Electrophoresis indicator

Bromophenol Blue 100mg

ddH2O 10mL

Electrophoresis Related Reagents

SDS Loading Buffer (2X)

For SDS-PAGE loading

0.5 M Tris-HCl，pH 6.8 2.5ml

Glycerol 2ml

10%SDS 4ml

0.1% Bromophenol Blue 0.5ml

ddH2O to 10mL

LDS Loading Buffer (4X)

For LDS-PAGE loading

Tris HCl 0.666g

Tris base 0.682g

LDS 0.8g

EDTA 0.006g

Glycerol 4g

SERVA Blue G250 (1% solution) 0.75ml

Phenol red (1% solution) 0.25ml

ddH2O to 10ml

Tris-Glycine SDS Electrophoresis Buffer (10X)

SDS-PAGE electrophoresis buffer

Tris base 29g

Glycine 144g

SDS 10g

ddH2O to 1000ml

Tris-Glycine Native Electrophoresis Buffer (10X)

Native PAGE electrophoresis buffer

Tris base 29g

Glycine 144g

ddH2O to 1000ml

MOPS SDS Electrophoresis Buffer (20X)

MOPS buffer system electrophoresis buffer

MOPS 104.6g

Tris base 60.6g

SDS 10g

EDTA 3g

ddH2O to 500ml

MES SDS Electrophoresis Buffer (20X)

MES buffer system electrophoresis buffer

MES 97.6g

Tris base 60.6g

SDS 10g

EDTA 3g

ddH2O to 500ml

Tricine SDS Electrophoresis Buffer (10X)

Tricine buffer system electrophoresis buffer

Tricine 179g

Tris base 121g

SDS 10g

ddH2O to 1000ml

Transfer Related Buffers

Tris-Glycine Transfer Buffer (25X)

Western transfer buffer

Tris base 18.2g

Glycine 90g

ddH2O to 500ml

Bis-Tis Transfer Buffer (20X)

Bis-Tris system transfer buffer

Bicine 10.2g

Bis-Tris (free base) 13.1g

EDTA 0.75g

ddH2O to 125ml

Blocking Buffers and Gel Recipes

5% Skim Milk Blocking Buffer

Western Blot blocking buffer

Skim milk powder 2.5g

TBST or PBST to 50ml

3% BSA Blocking Buffer

Western Blot blocking buffer

BSA 1.5g

TBST or PBST to 50ml

Resolving Gel Recipe

SDS-PAGE resolving gel

Solution 6%/10%/12%/16%/20%

50% Acrylamide/Bis 3/5/6/8/10 ml

Resolving gel buffer 9.4 ml

10%SDS 250 ul

50% Sucrose 4 ml

ddH2O 7.8/5.8/4.8/2.7/0.75 ml

TEMED 6.25 ul

APS 625 ul

Add TEMED and APS last, and mix thoroughly before pouring the gel.

50% Acrylamide/Bis

Gel stock solution

Acrylamide 48.3g

Bis 1.7g

ddH2O to 100ml

Resolving Gel Buffer

Resolving gel buffer

Tris 30.3g

HCl Adjust pH to 8.8

ddH2O to 250ml

50% Sucrose

Sucrose solution

Sucrose 50g

ddH2O to 100ml

Ammonium Persulfate (APS) (prepare fresh)

Gel polymerization initiator

Ammonium persulfate 100mg

ddH2O to 2ml

4% Stacking Gel

SDS-PAGE stacking gel

50% Acrylamide/Bis 1ml

Stacking gel buffer 4.2ml

10%SDS 125ul

ddH2O 6.3ml

TEMED 5ul

APS 1ml

Add TEMED and APS last, and mix thoroughly before pouring the gel.

Stacking Gel Buffer

Stacking gel buffer

Tris 11.4g

HCl Adjust pH to 6.8

ddH2O to 250ml