Griffin:Antibody Related Solutions & Recipes

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 228: Line 228:
*50 mM NaCl
*50 mM NaCl
*0.1% (v/v) Tween-20
*0.1% (v/v) Tween-20
 +
 +
==TE Buffer==
 +
 +
[http://openwetware.org/wiki/TE_buffer TE Buffer]
 +
 +
* 10 mM Tris, bring to pH 7.5 with HCl
 +
* 1 mM EDTA
 +
 +
pH is usually adjusted to 7.5 for RNA and 8.0 for DNA. The respective DNA and RNA nucleases are supposed to be less active at these pH values, but pH 8.0 can safely be used for storage of both DNA and RNA.
==Transfer Buffer==
==Transfer Buffer==

Revision as of 18:16, 20 January 2009

Contents

Acrylamide Stock Solution

To make 1L:

  • 30% Acrylamide: 300 g Acrylamide
  • 0.8% Bis-Acrylamide: 8.0 g Bis-Acrylamide

Materials:

  • Acrylamide (crystalline; store @ 4C)
  • Bisacrylamide (crystalline; store at room temperature)
  • Filter Unit (Nalgene 500 ml 0.2 micron disposable filter unit)
  • Light proof container

Methods: To make 500 ml Stock Solution

I) Place 300 ml ddH20 in beaker with stir bar

II) Add 150.0 g acrylamide to the H20 while stirring

III) Add 4.0 g bisacrylamide to the H20 while stirring

IV) Allow the acrylamide & bisacrylamide to dissolve

V) Bring the final volume to 500 ml

VI) Vacuum filter the acrylamide/bisacrylamide solution

VII) Place the acrylamide stock solution in a light proof container @ 4C

Blocking Buffer (Bovine Serum Albumin)

To make 1L:

  • 20 mM Tris: 2.42 g Tris Base pH 7.4 w/HCl
  • 150 mM NaCl: 8.76 g NaCl
  • 0.01% Tween-20: 0.1 ml Tween-20
  • 3% BSA: 30.0 g BSA (Fraction V)
  • 0.02% NaN3: 10.0 ml 2% NaN3

Antibody Reconstitution

For primary and secondary antibodies that undergo conjugation to detection molecules or solid phase, specific buffer reconstitution is critical for stability and shelf life.

Horseradish Peroxidase

primary and secondary antibodies: 0.6xPBS, 40% (v/v) glycerol, 1% BSA, 0.0067% thimerosal

Thimerosal is necessary because sodium azide is an irreversible inhibitor of HRP. Thimerosal contains 49.6% mercury. Mercury is strictly prohibited from entering wastewater. Although a single solution has a low concentration, mercury is bioaccumulated by algae and bacteria in drain pipes. It is this bioaccumulation and the continued disposal of mercury in drains that can potetntially contribute to disposal violations.

Alkaline Phosphatase

(primary and secondary antibodies) 0.5xPBS, 50% (v/v) glycerol, 1mM ZnCl2, 1mM MgCl2, 0.02% sodium azide

Biotin

(primary and secondary antibodies) 1xPBS, 1% BSA, 0.02% sodium azide

Flurochromes

(ie FITC, TR, CY, Alexa) (primary and secondary antibodies) 1xPBS, 1% BSA, 0.02% sodium azide

Protein A/G/L

(and agarose conjugates) 1xPBS, 0.02% sodium azide

Control IgG

1xPBS, 0.2% gelatin, 0.1% sodium azide (Same as unconjugated primary antibodies)

Control Sera

0.02% thimerosal

Blocking Buffer (milk)

To make 1L:

  • 20 mM Tris: 2.42 g Tris Base pH 7.4 w/HCl
  • 150 mM NaCl: 8.76 g NaCl
  • 0.01% Tween-20: 0.1 ml Tween-20
  • 5% Milk: 50.0 g Nonfat Dry Milk
  • 0.02% NaN3: 10.0 ml 2% NaN3

Casein solution

1. Add an appropriate amount of Hammersten grade casein to the buffer in a glass beaker with a magnetic spin bar. (Recommended concentration is 0.5%; maximum soluble casein is 1%.)

2. Place the beaker on a magnetic stirrer/hot plate.

3. Stir thoroughly while heating until the solution becomes translucent. Avoid foaming.

4. Remove the beaker from heat and let the solution cool. In some cases, it may also be desirable to filter the solution with a 0.45 to 1.2 µm pore size filter. For prolonged storage of casein solutions, refrigeration and preservatives (ThimerosalTM) are recommended.

Induction Conditions

Preparation of Nuclear Extract Inductions and Whole Cell Lysates

Anisomycin

0.25 mg/ml overnight

CoCl2

75 micromolar CoCl2 for 4 hours at 37C

EGF

5ng/ml for 4 hours at 37C

Etoposide

Solid etoposide is weighed out and dissolved in DMSO to a concentration of 68 mM. Cells are induced at 68 mM etoposide for 6 hours.

GM-CSF

5 minutes at 25ng/ml

γIFN

15 minutes at 200 units/ml

  • Include fetal calf serum when inducing with TNF-α and γ-interferon.

Heat Shock

39C for 5 hours

IL-6 treatment of NIH/3T3

grow up cells and add 30ng/ml of IL-6 for 15 minutes. Then, lyse cells with RIPA buffer to get WCL.

LPS/PMA

0.005 mg/ml final concentrations for LPS, 40ng/ml for PMA, induction time is 2 hours

PDGF

50 ng/mL for 5 minutes. Incubation time can be up to 30 minutes for successful induction

Phorbol

PMA or TPA; use at 40ng/ml for 2 hours at 37C.

  • PMA available from Gibco, cat# 13139-019

Serum Starved

no serum for 2 hours

Serum Starved + Serum - 1) no serum for 2 hours 2) add serum back for 15 minutes

TGFβ

10 ng/ml for 1 hour at 37 degees celcius.

TNFα

5 minutes at 10ng/ml

  • Include fetal calf serum when inducing with TNF-α and γ-interferon.

UV Irradiated

20 minutes under a UV light

NET-G Buffer

  • 150 mM NaCl
  • 5 mM EDTA
  • 50 mM Tris
  • 0.05% Triton X-100
  • 0.25% Gelatin (bovine skin, type III, approx. 225 bloom)
  • pH 7.4

NETN Buffer

Cell protein extraction buffer

  • 125 mM NaCl
  • 1 mM EDTA
  • 20 mM Tris-HCl pH 8.1
  • 0.5% Nonidet P-40
  • 10% Glycerol
  • 1X Protease Inhibitor Cocktail

RSB-100 Buffer

  • 100 mM Tris-HCl pH 7.4
  • 100 mM NaCl
  • 2.5 mM MgCl2
  • 40 ug/ml digitonin

RSB-100T Buffer

  • 100 mM Tris-HCl pH 7.4
  • 100 mM NaCl
  • 2.5 mM MgCl2
  • 40 ug/ml digitonin
  • 0.5% Triton X-100

Resolving Gel Buffer

To make 1L:

  • 0.75 M Tris pH 8.9: 90.8 g Tris Base pH 8.9 w HCl
  • 4 mM EDTA: 8.0 ml of 0.5 M EDTA (tetra Na is the most soluble)
  • 0.2% SDS: 20.0 ml 10% SDS

Running Buffer

To make 1L:

  • 50 mM Tris: 6.06 g Tris Base
  • 380 mM Glycine: 28.5 g Glycine
  • 1.6 mM EDTA: 0.67 g EDTA (tetra Na is the most soluble)
  • 0.1% SDS: 1.0 g SDS

Stacking Gel Buffer

To make 1L:

  • 0.1 M Tris pH6.7: 12.2 g Tris Base pH 6.7 w/H3PO4
  • 4 mM EDTA: 8.0 ml of 0.5 M EDTA (tetra Na is the most soluble)
  • 0.2% SDS: 20 ml 10% SDS

Stripping Buffer

To make 1L:

  • 62.5 mM Tris pH 6.8: 7.0 g Tris base ph 6.8 w/HCl
  • 100 mM 2-Mercaptoethanol: 7.0 ml 2-Mercaptoethanol (14.3 mol/L Stock)
  • 2% SDS: 20.0 g SDS

TENT Buffer

Tris, EDTA, NaCl, Triton: A mild lysis buffer that is also suitable for dialysis applications

Option 1

  • 50 mM Tris/HCl, pH 7.0
  • 5.0 mM EDTA
  • 150 mM NaCl
  • 0.05% (v/v) Tween-20

Option 2

  • 40 mM Tris-HCl, pH 8.8
  • 1.0 mM EDTA
  • 50 mM NaCl
  • 0.1% (v/v) Tween-20

TE Buffer

TE Buffer

  • 10 mM Tris, bring to pH 7.5 with HCl
  • 1 mM EDTA

pH is usually adjusted to 7.5 for RNA and 8.0 for DNA. The respective DNA and RNA nucleases are supposed to be less active at these pH values, but pH 8.0 can safely be used for storage of both DNA and RNA.

Transfer Buffer

To make 1L:

  • 25 mM Tris: 3.03 g Tris Base
  • 192 mM Glycine: 14.41 g Glycine
  • 0.02% SDS: 0.2 g SDS
  • 20% Methanol: 200 ml Methanol

Tris Buffer Saline (TBS Tween, Wash Buffer)

To make 1L:

  • 20 mM Tris: 2.42 g Tris Base pH 7.4 w/ HCl
  • 150 mM NaCl: 8.76 g NaCl
  • 0.01% Tween-20: 0.1 ml Tween-20 and and
Personal tools