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. Various components can be combined to maximize the shelf life of an antibody in solution.
Bovine Serum Albumin
BSA is a stabilizer protein used in peptides and antibody solutions to mitigate protein aggregation. BSA is used because of its stability, lack of effect in many biochemical reactions, and its low cost since large quantities of it can be readily purified from bovine blood, a byproduct of the cattle industry.
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.
Glycerol
Glycerol is a colorless, odorless, viscous liquid. Glycerol is a common component of solvents for enzymatic reagents stored at temperatures below zero degrees Celsius since the solution will remain viscous and able to pipet.
Sodium Azide
Sodium azide is an ionic chemical compound with the formula NaN3. This colorless azide salt is added to prevent bacterial contamination; sodium azide acts as a bacteriostatic by inhibiting cytochrome oxidase in gram-negative bacteria. Will inactivate HRP.
Thimerosal
Thimerosal is an organomercury compound (approximately 49% mercury by weight) used as an antiseptic and antifungal agent in solution where it is prohibitive to use NaN3.
Unconjugated primary antibody solutions
Standard
- 0.2 ug/ul in 1X PBS, 0.2% gelatin, 0.1% sodium azide ; Store at 4C
Concentrated ChIP & EMSA grade
Also known as 'transcruz' or 'X' form
- 2.0 ug/ul in 1X PBS, 0.1% sodium azide ; Store at 4C or -20C (aliquot if frozen)
In vitro/biological study grade
- 2.0 ug/ul in filter sterilized 1X PBS
Conjugated primary antibody solutions
Horseradish Peroxidase
- primary and secondary antibodies: 0.6X PBS, 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) 0.5X PBS, 50% (v/v) glycerol, 1mM ZnCl2, 1mM MgCl2, 0.02% sodium azide
Biotin
- (primary and secondary antibodies) 1X PBS, 1% BSA, 0.02% sodium azide
Flurochromes
- (ie FITC, TR, CY, Alexa) (primary and secondary antibodies) 1X PBS, 1% BSA, 0.02% sodium azide
Agarose
- (Protein A/G/L and CnBr agarose conjugates) 1X PBS, 0.02% sodium azide
Control solutions
Control IgG
- 1X PBS, 0.2% gelatin, 0.1% sodium azide (same as unconjugated primary antibodies)
Control Sera
- Add 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.
Coomassie Stain
- Dissolve 2g Coomassie Blue (Serva Blau) in 250ml water
- Slowly add 75ml of glacial acetic acid
- Add 500ml of ethanol
- q.s. to 1000ml with water
Final concentrations: 0.2% Coomassie Blue 7.5% Acetic Acid 50% Ethanol
Destain:
- methanol 20%: 800 mL
- glacial acetic acid 5%:200 mL
- H2O 75%: 3 L
DEPC H20
Add 0.1% DEPC to H20
Shake well and Autoclave
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
PBS
Phosphate buffered saline (1x PBS):
- 9.1 mM dibasic sodium phosphate
- 1.7 mM monobasic sodium phosphate
- 150 mM NaCl
Adjust pH to 7.4 with NaOH
Ponceau S
Ponceau S dye is used to make a stain for rapid reversible staining of protein bands on nitrocellulose or PVDF membranes (the ones we use) and also for staining proteins on nitrocellulose acetate membranes. The following are 2 common stains formulations
2% Ponceau S
2% Ponceau S (w/v) in 30% TCA, 30% sulfosalicylic acid
- 2g Ponceau S
- 30g Trichloracetic acid
- 30g Sulfosalyclic acid
- H20 to 100ml
0.1% Ponceau S
0.1% Ponceau S (w/v) in 5% acetic acid
- 5ml glacial acetic acid
- 90ml deionized water
- 5mg (0.1%) Ponceau S powder
- Stain can be reused multiple times.
PMSF stock solution
Mass: 174.194
0.1 M PMSF stock: 1.0 g PMSF + 57 ml isopropanol, store at -20°C
- Soluble to 10 mg/ml in isopropanol, ethanol, methanol,and 1,2-propanediol.
- Suggested working concentration: 17-174 ug/ml (100-1000uM).
- Stability: Dry crystals are stable at room temperature. Stable at least nine months at +25 degC in 100% isopropanol.
PMSF stability in aqueous buffer
The half-life of 100 uM PMSF at 25 C (measured as ability to inhibit chymotrypsin) is 110 min. at pH 7.0 (phosphate buffer), 55 min. at pH 7.5 (Hepes), and 35 minutes at pH 8.0 (Tris). At 4 C, 100 uM PMSF is completely inactivated in 30h at pH 7.6 (Hepes), 22h at pH 8.0 (Tris), and 10h at pH 8.6 (Tris). Presence of EDTA and other metal chelators, DTT or 2-mercaptoethanol does not interfere with the action of PMSF.
PMSF safety precaution
DMSO solvent is haxardous for solubility of PMSF (acetylcholine esterase inhibitor). DMSO can penetrate skin as a vehicle to carry dissolved substances with it. Isopropanol is a good choice, being affordable, low toxicity and less polar than MeOH or EtOH, so PMSF is more stable in this solvent.
1.0 M PMSF stock: 0.87 g PMSF + 5 ml DMSO, store at -20°C
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
20% SDS
Sodium Laurel Sulfate or Sodium Dodecyl: Stock Solution
- SDS (Electrophoresis-grade): 200.0 g
- Deionized H2O: 800 ml
- Total Volume: 1000.0 ml
- After 800 ml of dH2O is added--stir, top off to 1000.0 ml. Do not pH.
- Heat to 68°C for dissolution.
- Aliquot 100 ml into (10) 200 ml square bottles
- Do not autoclave.
2.5X Sample Buffer (Laemmli sample buffer; LSB)
- 25ml Glycerol
- 12.5g DTT
- 7.5g SDS
- 15.6ml 1M Tris-HCl pH7.5
- 5mg Bromophenol Blue
- H20 to 100ml
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
1M Tris-HCl
- 141g TRIS base in 800ml H20
- pH to 7.5
- H20 to 1 Liter
Wortmannin
Solubility: Soluble in DMSO or ethanol; very poorly soluble in water; maximum solubility in plain water is estimated to be about 10-50 µM
Wortmannin (M.W. 428.43) dissolve in dimethyl sulfoxide (DMSO) to a concentration of 50 mM as a stock solution.