Buffers

Urea Lysis Buffer (Xiaomin & Todd’s recipe)

 * 8M Urea
 * 4% CHAPS
 * 50mM DTT or 10% b-ME
 * 40mM Tris pH 8.0
 * 2.5mM EDTA

Lysis Buffer (Yuan Lab)
Basic Lysis Buffer Working Lysis Buffer
 * 25ml 1M HEPES (pH 7.5) [50mM]
 * 15ml 5M NaCl [150mM]
 * 1ml 500mM EDTA [1mM]
 * 2.5ml 500mM EGTA [2.5mM]
 * 456.5ml ddH2O
 * One tablet of protease inhibitor cocktail in 1ml dH2O
 * 1ml 1M NaF (100X)
 * 100ul 1M DTT (1000X)
 * 100ul 1M SV (sodium orthovanadate, Na3VO4, 920mg in 5ml H2O) (1000X)
 * 1ml 100mM PMSF (174mg phenylmethylsulfonyl fluoride in 10 ml Isopropanol) (100X)
 * Add to 100ml basic lysis buffer
 * Store at -20°C
 * For extra phosphatase inhibiton add 50'ml NaF (Tyr Kinase inhibitor) and 10'ml SV (Ser/Thr Kinase inhibitor)
 * For lysing: add 1% NP-40, 0.1% SDS for RIPA or 1% TritonX-100 for IP/GST pulldown

10X Running Buffer (for homemade PAGE)

 * 188g Glycine
 * 30.2g Tris-base
 * 50ml 20% SDS
 * 1L dH­2O

Transfer Buffer (1L)

 * 5.82g Tris-base
 * 2.93g Glycine
 * 100ml MeOH

· Alternative (Yuan lab) for 2L
 * 29g Glycine
 * 58g Tris-base
 * 18.5ul 20% SDS
 * 200ml MeOH

TBST (2L)

 * 60ml 5M NaCl
 * 50ml 1M Tris-HCl (pH 7.5)
 * 10ml 20% Tween-20
 * 3ml KCl2

PBST (2L)

 * 18g NaCl
 * 0.4g KCl
 * 2.88g Na2HPO4
 * 0.68g KH2PO4
 * 1ml Tween-20

1.5M Tris (pH 8.8)

 * 36.9g Tris (HCl)


 * 153.9g Tris-base


 * 1L H2O

10% APS

 * 1g ammonium persulfate in 15ml falcon tube


 * Fill to 10ml with dH2O

10X PBS

 * 160g NaCl
 * 4g KCl
 * 28.8g Na2HPO4
 * 4.8g KH2PO4
 * Bring up to 2L with dH2O
 * pH at 7

Ampicillin 1000X (100mg/ml)

 * 1g ampicillin
 * 4.3ml EtOH
 * 5.7ml dH2O

2.5M CaCl2

 * 183.7g CaCl2 dihydride (Sigma, tissue culture grade)
 * H2O to 500ml
 * Filter sterilize through 0.45um filter
 * Store a -20°C in 10ml aliquots

2X HEPES

 * 11.9g HEPES acid
 * 0.21g Na2HPO4
 * 800ml H2O
 * Titrate to pH 7.05 (7.05-7.12) with 5N NaOH
 * Add H2O to 1L
 * Filter sterilize through 0.45mm filter
 * Store at -20°C in 50ml aliquots

Triton Extraction Buffer
In PBS:
 * 0.5% Triton X-100
 * 2mM PMSF (from 100mM stock)
 * 0.02% NaN3 (from 1% stock)


 * Can add 5mM sodium butyrate to preserve acetylation marks

NuPAGE® MOPS SDS Running Buffer
The NuPAGE® MOPS SDS Running Buffer (20X) is available from Invitrogen


 * 50 mM MOPS
 * 50 mM Tris base
 * 0.1% SDS
 * 1 mM EDTA
 * pH 7.7

To prepare 500 ml of 20 X NuPAGE® MOPS SDS Running Buffer, dissolve the following reagents to 400 ml ultrapure water:
 * MOPS 104.6 g
 * Tris Base 60.6 g
 * SDS 10 g
 * EDTA 3.0 g

Mix well and adjust the volume to 500 ml with ultrapure water.

Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

For electrophoresis, dilute this buffer to 1X with water. The pH of the 1X solution is 7.7. Do not use acid or base to adjust the pH.

NuPAGE® MES SDS Running Buffer
The NuPAGE® MES SDS Running Buffer (20X) is available from Invitrogen


 * 50 mM MES
 * 50 mM Tris base
 * 0.1% SDS
 * 1 mM EDTA
 * pH 7.3

To prepare 500 ml of 20 X NuPAGE® MES SDS Running Buffer, dissolve the following reagents to 400 ml ultrapure water:


 * MES 97.6 g
 * Tris Base 60.6 g
 * SDS 10 g
 * EDTA 3.0 g

Mix well and adjust the volume to 500 ml with ultrapure water.

Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

For electrophoresis, dilute this buffer to 1X with water. The pH of the 1X solution is 7.3. Do not use acid or base to adjust the pH.

NuPAGE® Tris-Acetate SDS Running Buffer
The NuPAGE® Tris-Acetate SDS Running Buffer (20X) is available from Invitrogen


 * 50 mM Tricine
 * 50 mM Tris base
 * 0.1% SDS
 * pH 8.24

To prepare 500 ml of 20 X NuPAGE® Tris-Acetate SDS Running Buffer, dissolve the following reagents to 400 ml ultrapure water:


 * Tricine 89.5 g
 * Tris Base 60.6 g
 * SDS 10 g

Mix well and adjust the volume to 500 ml with ultrapure water.

Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

For electrophoresis, dilute this buffer to 1X with water. The pH of the 1X solution is 8.24. Do not use acid or base to adjust the pH.

NuPAGE® Transfer Buffer
The NuPAGE® Transfer Buffer (20X) is available from Invitrogen


 * 25 mM Bicine
 * 25 mM Bis-Tris (free base)
 * 1 mM EDTA
 * pH 7.2

To prepare 125 ml of 20 X NuPAGE® Transfer Buffer, dissolve the following reagents to 100 ml ultrapure water:


 * Bicine 10.2 g
 * Bis-Tris (free base) 13.1 g
 * EDTA 0.75 g

Mix well and adjust the volume to 125 ml with ultrapure water.

Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

For western transfer, dilute this buffer to 1X with water. The pH of the 1X solution is 7.2. Do not use acid or base to adjust the pH.

NuPAGE® LDS Sample Buffer
The NuPAGE® LDS Sample Buffer (4X) is available from Invitrogen


 * 106 mM Tris HCl
 * 141 mM Tris base
 * 2% LDS
 * 10% Glycerol
 * 0.51 mM EDTA
 * 0.22 mM SERVA® Blue G250
 * 0.175 mM Phenol Red
 * pH 8.5

To prepare 10 ml of 4 X NuPAGE® LDS Sample Buffer, dissolve the following reagents to 8 ml ultrapure water:
 * Tris HCl 0.666 g
 * Tris Base 0.682 g
 * LDS 0.800 g
 * EDTA 0.006 g
 * Glycerol 4 g
 * SERVA® Blue G250 (1% solution) 0.75 ml
 * Phenol Red (1% solution) 0.25 ml

Mix well and adjust the volume to 10 ml with ultrapure water.

Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

For electrophoresis, prepare your samples in this buffer as described. The pH of the 1X solution is 8.5. Do not use acid or base to adjust the pH.

Tris-Glycine Native Sample Buffer
The Tris-Glycine Native Sample Buffer (2X) is available from Invitrogen


 * 100 mM Tris HCl
 * 10% Glycerol
 * 0.0025% Bromophenol Blue
 * pH 8.6

To prepare 10 ml of 2 X Tris-Glycine Native Sample Buffer, mix the following reagents:
 * 4 M Tris HCl 4 ml
 * 10% Glycerol 2 ml
 * 0.1% Bromophenol Blue 0.5 ml
 * Deionized Water 3.5 ml

Mix well and adjust the pH of the solution is 8.6.

Store at +4° C. The buffer is stable for 6 months when stored at +4° C.

Use this buffer to prepare samples for non-denaturing NuPAGE® Tris-Acetate gel electrophoresis.

Tris-Glycine Native Running Buffer
The Tris-Glycine Native Running Buffer (10X) is available from Invitrogen


 * 25 mM Tris base
 * 192 mM Glycine
 * pH 8.3

To prepare 1000 ml of 10 X Tris-Glycine Native Running Buffer, dissolve the following reagents in 900 ml deionized water:
 * Tris Base 29 g
 * Glycine 144 g

Mix well and adjust the volume to 1000 ml with ultrapure water.

Store at room temperature. The buffer is stable for 6 months when stored at room temperature.

For native electrophoresis, dilute this buffer to 1X with water The pH of the 1X solution is 8.3. Do not use acid or base to adjust the pH.

5M NaCl

 * 584g NaCl
 * que to 2L H2O

50X TAE

 * 484g tris base
 * 114mL acetic acid
 * 74g EDTA
 * que to 2L H2O