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

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.

Specifications for use with research antibodies

US origin or USDA certified
Fraction V powder
Heat-shock treated
Protease free
DNAse free
RNAse free
IgG free
fatty acid free

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. Glycerol displaces water in solution and minimizes protein conformational movements.

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

Type B calf gelatin, 100 Bloom

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 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 (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

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

Carbonate Buffer

To make 1L of 50 mM pH 8.2-9.6

Sodium carbonate (105.99 g/mol): 5.3 g
pH should start around pH 8.2 +/- use 5M NaOH to increase the pH of this buffer to 9.6
Optional add 0.02% 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.

Cell Culture Solution Preparations

Make sure that the culture media contains an antibiotic (penn/strep); this alone will prevent the majority of contamination issues.
Dissolve small molecule solutes in either 100% ethanol or 100% DMSO. These solvents are not conducive to bacterial or fungal growth, and using them will prevent contaminants from multiplying in a stock solution.

Sterile Filtration

Small molecules can be sterile filtered through a 0.22 µm filter. There will be some loss of the product to the filter. Sterile filtering is the best precaution that can be taken against contamination; however the loss of material to the filter can introduce some variability into the experiment as well.

Coomassie Stain

Coomassie Dye:

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

Coupling Ratios (F:P)

3 to 8 biotin per IgG molecule (biotin MW: 244)
4 to 7 FITC per IgG molecule (FITC MW: 390)
1 to 2 HRP per IgG molecule (HRP MW: 40,000)
1 APC per IgG molecule (APC MW: 100,000)
~1 PE per IgG molecule (PE MW: 240,000)
AF405: 1-3 moles of dye: mole of IgG
AF488: 4-9 moles of dye: mole of IgG
AF647: 3-7 moles of dye: mole of IgG

DEPC H20

Add 0.1% DEPC to H20

Shake well and Autoclave

DMSO Vehicle control

Dimethyl sulfoxide (DMSO) is a clear water soluble liquid that can dissolve both polar and nonpolar compounds.

Chemicals that are insoluble in water may require DMSO as an ideal solvent for cell permeable drugs. The disadvantage is that DMSO can affect cells, and so there must always be a parallel control setting where cells undergo treatment with everything but the chemical; also known as a 'vehicle' control. The treatment group of cells will then be known with confidence to be chemical-dependent relative to the vehicle-dependent negative control.

DMSO for cell culture should ideally be <0.1% (avoid use greater than 1% DMSO), since DMSO is cytotoxic at higher concentrations. However this is not always realistic depending on the chemical inhibitor. Determining a vehicle (DMSO) dose/response curve (viability) can determine the best approach. Hydrophobic/polar compounds may only dissolve in DMSO, so up to 5% DMSO in the culture assay may be necessary, in which case the vehicle control is essential.

Precipitation of the inhibitor out of the DMSO solution when it is pipetted into aqueous media is a characteristic of some DMSO soluble inhibitors. DMSO soluble inhibitors that are prone to precipitation can undergo dilution into aqueous media, however the media should contain 0.1-0.5% DMSO before the inhibitor is added, and they must be mixed very quickly to avoid precipitation.

Ideal maximum amount of DMSO relative to the media is 0.1% since DMSO alone can have significant side effects. The % of DMSO that is cytotoxic to cells is generally 0.1% and above. Final concentration of DMSO ideally should not exceed 0.5%.

Epitope tag sequences

FLAG epitope

5' GAC TAC AAG GAC GAC GAT GAC AAG 3'

3' CTG ATG TTC CTG CTG CTA CTG TTC 5'

D Y K D D D D K

HA epitope

5' TAT CCG TAT GAT GTT CCT GAT TAT GCT AGC CTC 3'

3' ATA GGC ATA CTA CAA GGA CTA ATA CGA TCG GAG 5'

Y P Y D V P D Y A S L

Omni epitope

MASMTGGQQMGRDLYDDDDKD

MASMTGGQQMGRDLYDDDDKV (invitrogen sequence)

FSGO (0.5% Fish Skin Gelatin Oil)

100 mL

1 ml Teleostein Gelatin ∼45% in ddH2O
0.5 ml 20% Triton X-100
10 ml 10X PBS
0.1 g Casein (Enzymatic Hydrolysate)
88.5 ml ddH2O

Induction Conditions

Preparation of Nuclear Extract Inductions and Whole Cell Lysates

Anisomycin

0.25 mg/ml overnight

Camptothecin

4.0 uM for 4 hours

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

αIFN

100 ng/ ml , 15 or 30 min

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

Staurosporine

0.25 ug/ml for 6-10 hours (8 hours)

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

Peptide Substrate

10 mM TRIS pH 7.5
150 mM NaCl
0.02% azide

Ponceau S

Ponceau S dye is used to make a stain for rapid reversible staining of protein bands on nitrocellulose or PVDF membranes, and for staining proteins on nitrocellulose acetate membranes. Stain can be reused multiple times. The following are 2 common formulations;

2.0% 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

PMSF stock solution

PMSF

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

Recombinant Protein Solvents

50 mM Tris, 2 mM EDTA, 250 mM NaCl, 250 mM NDSB : Standard dry down buffer for lyophilized recombinant protein.

1X PBS, 50% Glycerol, 5 mM DTT : Standard solvent for solutions of recombinant protein.

Store any thawed aliquot in refrigeration at 2° C to 8° C for up to four weeks, and any frozen aliquot at -20° C to -80° C for up to one year. It is recommended that frozen aliquots be given an amount of standard cryopreservative (such as Ethylene Glycol or Glycerol 5-20% v/v), and refrigerated samples be given an amount of carrier protein (such as heat inactivated FBS or BSA to 0.1% v/v) or non-ionic detergent (such as Triton X-100 or Tween 20 to 0.005% v/v), to aid stability during storage.

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

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

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.

Sample Buffer for protein loading

Premade SCBT

Electrophoresis sample buffer (2X): 100mM 2-(N-Morpholino)- ethanesulfonic acid(MES), 10 mM Na EDTA, 15% glycerol, 1.5% SDS, 0.3% Triton X-100, 25mM TCEP-HCL, 7.5 mM DTT, 0.0025% Bromophenol Blue. Available pre-made (sc-24945).

2.5X (Laemmli sample buffer; LSB)

25ml Glycerol = 25%
12.5g DTT = 12.5%
7.5g SDS = 7.5%
15.6ml 1M Tris-HCl pH7.5 =
5mg Bromophenol Blue = 0.005%
dH20 to 100ml

2X SDS sample buffer

4g SDS = 4%
2ml 2-Mercaptoethanol = 2%
20ml of 100% glycerol = 20%
1.36g TRIS base (121.1) = 112.5mM
10 mg Bromophenol blue = 0.01%
dH20 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.