Griffin:Antibody Related Solutions & Recipes
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.
Gelatin
BLOOM NUMBER:
Bloom number is an indication of the strength of a gel formed from a solution of known concentration. The Bloom unit is a measure of the force (weight) required to depress a given sample area of gel a distance of 4 mm; the higher the Bloom number, the stronger the gel.
Bloom number is proportional to the average molecular weight:
Bloom Number Average Molecular Weight
50 - 125 (Low Bloom) 20,000 - 25,000
175 - 225 (Medium Bloom) 40,000 - 50,000
225 - 325 (High Bloom) 50,000 - 100,000
Gelatin is a heterogeneous mixture of water-soluble proteins of high average molecular weights, present in collagen. The proteins are extracted by boiling skin, tendons, ligaments, bones, etc. in water. Type A gelatin is derived from acid-cured tissue and Type B gelatin is derived from lime-cured tissue.1 Gelatin is used as a stabilizer, thickener and texturizer in foods; in the manufacture of rubber substitutes, adhesives, cements, lithographic and printing inks, plastic compounds, artificial silk, photographic plates and films, matches, and light filters for mercury lamps; in textiles; to inhibit crystallization in bacteriology and prepare cultures; in PCR hybridization in molecular biology; in the pharmaceutical industry as a suspending agent, encapsulating agent and tablet binder; and in veterinary applications as a plasma expander and hemostatic sponge.
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
- 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