Haematologic Technologies, Inc. - Frequently Asked Questions

Our "Technical Notes" section is not meant to serve as a methods section, but instead focuses on some of the most common questions that we receive in our technical services department. Many of the recommendations that follow are generalized, and may differ from many established protocols. If you have technical questions that we do not address in this section, or if you require further assistance, please do not hesitate to call (802) 878-1777 and ask for our technical services department.

PRODUCT STORAGE AND HANDLING (Back to Tech Notes)
All orders shipped from Haematologic Technologies, Inc. are accompanied by product information sheets which describe proper storage conditions. In order that we may warrant product stability, it is imperative that these storage conditions be maintained at all times. Many of our protein preparations are formulated in 50% (vol/vol) glycerol/H₂O which will remain in fluid phase during storage at -20^oC. This preferred method of storage yields the greatest protein stability while still allowing access to the stock protein sample without repeated thawing and freezing steps. For some of our new customers who may not be accustomed to handling proteins in glycerol/H2O, here are a few basic tips:

All products which are formulated with either glycerol/H2O or aqueous buffer are delivered in microcentrifuge tubes. By briefly centrifuging the samples in their original containers, complete recovery of the sample at the bottom of the tube will be accomplished.
All products which are formulated with glycerol/H2O should be stored at -20^oC. Temperatures lower than -30^oC should be avoided in order to prevent a phase transition.
When preparing to make a dilution of the stock sample, remove the sample from storage at -20^oC and place on ice for a brief period of time (5-10 min). The sample will become less viscous and thus easier to pipette.
Never allow protein solutions to remain at room temperature for excessive periods of time. Elevated temperatures may enhance the rate of protein degradation.
Avoid storing or maintaining dilute protein samples for a long period of time. In general, purified proteins are inherently more stable in concentrated form.
Many proteins are "sticky" by nature. To avoid losing protein due to adsorption, extremely dilute protein samples should be prepared in buffers containing excipients such as bovine serum albumin, poly(ethylene glycol), or gelatin.

ENZYME ASSAYS AND ACTIVITY UNITS (Back to Tech Notes)
Data sheets: Data sheets documenting the specific activity of HTI products are included in all shipments. The type of assay performed and an explanation of the units of activity are included in footnotes.

Assays and units: In general, the activities of HTI products are measured in standard PT and APTT based clotting assays where one unit of activity is defined as the amount of activity present in 1 ml of normal pooled human plasma. Exceptions to this rule include the following:

NIH Units: Thrombin Activity is reported in NIH units. This activity is determined by comparison to U.S. Standard Thrombin, Lot J.

Chromogenic Substrate Hydrolysis: The activity of some enzymes (ex. APC, plasmin) are determined by the rate of hydrolysis of chromogenic substrates. 1 unit of activity is defined as the hydrolysis of 1 µmole of substrate per minute, under defined reaction conditions. It should be noted that this activity is highly dependent on the particular substrate used, the concentration of substrate, pH, ionic strength and temperature. The conditions are specified on the product data sheet included in all shipments.

WESTERN BLOTTING (Back to Tech Notes)

THE RECOMMENDATIONS BELOW REPRESENT GENERAL GUIDELINES AND ARE NOT MEANT TO BE ABSOLUTES. OPTIMIZATION OF ASSAY CONDITIONS IS THE RESPONSIBILITY OF THE RESEARCHER.

Basic buffers:

TBS: 8.76 grams of NaCl and 2.42 grams of Tris base in 900 ml of deionized water. Adjust pH to 7.4 with 6N HCl. Adjust the volume to 1 liter with deionized water.

PBS: 8.0 grams of NaCl, 0.2 grams of KCl, 1.44 grams of Na2HPO4 and 0.24 grams of KH2PO4 in 900 ml of deionized water. Adjust pH to 7.4. Adjust volume to 1 liter with deionized water.

Blocking buffer: TBS or PBS containing one of the following: 2%(w/v) BSA, ovalbumin or casein, or 5%(w/v) non-fat powdered milk.
BSA/Tween buffer: TBS or PBS containing 0.02%(w/v) BSA and 0.05% (v/v) Tween 20.

Blocking conditions: After the protein samples have been transferred from the gel to either a nitrocellulose or PVDF membrane, remaining binding sites are blocked by incubating the blot in blocking buffer for 2 hours at room temperature or overnight at 4oC. Be sure to gently mix or agitate the blot during all blocking and incubation steps.

Wash steps: Wash the blot at least three times (incubating for 3 minutes with each wash) with BSA/Tween buffer between each procedural step.

Primary antibody: Primary antibodies should be diluted in BSA/Tween buffer. Dilute monoclonals and affinity purified polyclonals to 5 micrograms per ml. Dilute non-affinity purified polyclonals to 25 micrograms per ml. Incubate the blot with the primary antibody for 2 hours at room temperature, or overnight at 4^oC. These dilutions are also recommended if your primary antibody is conjugated. Further optimization of these conditions may be necessary for your application.

Secondary antibody: Use a peroxidase conjugated secondary antibody that is appropriate for your primary antibody (i.e., if you are using a murine IgG monoclonal, you may select a peroxidase conjugated goat anti-mouse IgG). Dilute the secondary antibody according to the manufacturer’s suggestions. Incubate with the blot for 1 to 2 hours at room temperature.

Development: Development may be done using the DAB (3,3'-diaminobenzidine) detection system (see Sigma catalog #D4418) or by chemiluminescence (see Amersham ECL Western Blotting Detection Reagents). Follow the manufacturer’s instructions for either application.

CLEAVAGE OF FUSION PROTEINS (Back to Tech Notes)
Conditions: Because of the subtle changes in secondary and/or tertiary structure which occur among different fusion proteins we cannot recommend a precise set of conditions that will optimize the factor Xa or thrombin cleavage of all fusion proteins. Instead, we offer some basic recommendations for starting conditions, and then suggest changes that may optimize the cleavage of your protein.

Enzymes: Bovine factor Xa (HTI catalog #BCXA-1060) is employed for removal of affinity tags from fusion proteins which contain a factor Xa recognition site. Human alpha-thrombin (HTI catalog #HCT-0020) is employed for removal of affinity tags from fusion proteins which contain a thrombin recognition site.

Concentrations: Start with a 1:50 molar ratio of enzyme to substrate (this is assuming that your substrate protein is in solution in the range of 1 to 50 micromolar). To make this calculation, you can use a molecular weight of 45,300 for bovine factor Xa, and 36,700 for both human or bovine thrombin.

Buffer choices: Thrombin and factor Xa are serine proteases that function best when the pH is between 7.0 and 8.5, and the ionic strength is near or equivalent to that of a 0.15M NaCl solution. TBS or HBS (20mM Tris (or Hepes), pH 7.5, containing 0.15M NaCl) are suitable buffers. For factor Xa, we also recommend including 2 mM CaCl2.

Temperature: Generally these experiments are done at room temperature, although elevated temperatures (i.e., 37^oC) which increase the hydrolysis rate will also work.

Optimization: To optimize the cleavage of fusion proteins a time-course experiment followed by SDS-PAGE analysis is often useful. Data from the time course experiment should be examined for both completeness of cleavage, as well as specificity. Generally reactions can be accelerated by increasing: a) the enzyme concentration; b) the temperature; and c) the pH (but not over pH 8.5), however an increased rate may also be accompanied by non-specific cleavage patterns.

IMMUNOHISTOCHEMISTRY (Back to Tech Notes)

THE RECOMMENDATIONS BELOW REPRESENT GENERAL GUIDELINES AND ARE NOT MEANT TO BE ABSOLUTES. OPTIMIZATION OF ASSAY CONDITIONS IS THE RESPONSIBILITY OF THE RESEARCHER.

Buffer:
HBS: 8.76 grams of NaCl and 4.8 grams of Hepes in 900 ml of deionized water. Adjust pH to 7.4 with 10 N NaOH. Adjust the volume to 1 liter with deionized water. For antibody/antigen interactions that are calcium dependent, include 2 mM CaCl2 in the buffer. The frequent need for calcium containing buffers is the reason we do not use PBS for this and many other applications.

Antibody: Start with an antibody concentration of 100 nM and then adjust this concentration to optimize your specific application.

Incubations: Recommended incubation times are 30 to 60 minutes at room temperature or overnight (16 hours) at 4^oC.Detection:For detection, we recommend using a FITC-labeled secondary antibody. These are readily available from a number of different suppliers, and we make no specific recommendations. Follow the manufacturer’s instructions for use of the FITC conjugate.

ELISA (Back to Tech Notes)

THE RECOMMENDATIONS BELOW REPRESENT GENERAL GUIDELINES AND ARE NOT MEANT TO BE ABSOLUTES. OPTIMIZATION OF ASSAY CONDITIONS IS THE RESPONSIBILITY OF THE RESEARCHER.

Basic buffers:
TBS: 8.76 grams of NaCl and 2.42 grams of Tris base in 900 ml of deionized water. Adjust pH to 7.4 with 6N HCl. Adjust the volume to 1 liter with deionized water.

Coating buffer: 50 mM Sodium Carbonate, pH 9.6. 1.7 grams of Na2CO3, 2.86 grams of NaHCO3 in 900 ml of deionized water. Check the pH to assure that it is 9.6 and adjust if necessary. Adjust the final volume to 1 liter using deionized water.

Blocking buffer: TBS containing 2.0% (w/v) BSA.

Wash buffer: TBS containing 0.05% (v/v) Tween 20

Assay buffer: TBS containing 0.02%(w/v) BSA and 0.05% (v/v) Tween 20.

Coating plates: Select assay plates that have been certified for use in ELISA applications. Dilute the coating protein (generally an antibody, however some competitive assay formats may require the antigen to be immobilized on the plate) to the desired concentration in coating buffer. For monoclonal antibodies and affinity purified polyclonal antibodies, we recommend a coating concentration of 10 micrograms per ml. For non-affinity purified polyclonals, we recommend a coating concentration of 25 micrograms per ml. Add the coating solution to the assay plate (0.1 ml per well is standard practice), cover the plate and allow it to incubate for 2 hours at room temperature, or overnight (approximately 16 hours) at 4oC.

Blocking plates: Remove the coating solution from the plate and fill the wells to 90% of their capacity (approximately 400 microliters for a standard 96 well plate) with blocking buffer. Incubate the plate for 2 hours at room temperature, or overnight (approximately 16 hours) at 4^oC.

Standards: Standard curves are typically generated by diluting the standard to 100 nanograms per ml in assay buffer, and then serially diluting to make eight standard concentrations in the range of 100 to 0.78 nanograms per ml. Apply 0.1 mls of each dilution to appropriate wells in the plate. The standard is typically run in duplicate.

Samples: Samples are diluted in assay buffer so that they will fall within the working range of the standard curve (0.78 to 100 nanograms per ml). Apply 0.1 mls of each sample to the appropriate wells in the plate. The samples are typically run in duplicate at two or more dilutions.

Incubation times: Primary incubations (i.e., those involving application of the standard or samples to the plate) are generally performed for either 2 hours at room temperature or overnight (16 hours) at 4^oC. Secondary incubations (those involving the application of secondary antibodies or antibody conjugates are generally done for 1 hour at room temperature. Incubation times may be further shortened by performing steps at 37^oC when possible. A dilution of 5 micrograms per mL for monoclonals and 10 micrograms per mL for polyclonals is recommended as a starting point. Further optimization may be necessary depending on your assay.

Development: The two most common enzymes utilized for detection are horseradish peroxidase and alkaline phosphatase. For horseradish peroxidase the most common substrates are tetramethylbenzidine (TMB) and o-phenylenediamine (OPD). The common substrate for alkaline phosphatase is p-nitrophenyl phosphate (PNPP). We recommend following the manufacturers instructions for the use of these substrates.

Optimization: Developing a sensitive and reproducible ELISA requires optimization of many conditions including choice of antibodies, antibody concentrations, incubation times, incubation temperatures, wash conditions, etc. The choice of antibody and the concentration of antibody used may affect both sensitivity and background noise. In addition, more (or less) stringent buffer conditions may be required to achieve optimal results.

FAQ (Tech Notes)

QUESTION #1) Most of your proteins are supplied in 50% glycerol/water at relatively high protein concentrations. May I dilute the protein samples out in a buffer of my choice for long term storage?

ANSWER #1) Our proteins are supplied in 50% glycerol/water (and may contain some residual ammonium sulfate; <0.1M). When stored at -20^oC the glycerol/water formulation will remain in an aqueous state. We have found this storage formulation to yield the greatest protein stability, while still allowing access to stock protein solutions without subjecting them to a potentially damaging freeze/thaw cycle.

The relatively high protein concentrations allow the user to make nearly 1000-fold dilutions of the stock samples to achieve physiologic protein concentrations for experimental applications. For applications which require much higher protein concentrations, or which may be sensitive to the presence of free amine groups, we recommend dialysis of the proteins prior to use.

HTI will warrant the stability of its proteins only under the original storage conditions. Therefore, we recommend re-formulation of the stock material only for immediate experimental purposes and not for long term storage purposes. If the proteins samples must be diluted and stored for applications such as ELISA standards, we recommend that excipients such as BSA or poly(ethylene glycol) be added to the buffer to help stabilize the protein and prevent loss due to adsorption to the storage vessel walls.

QUESTION #2) Your data sheets for proteins which are supplied in the 50% glycerol/water formulation indicate that they are to be stored at -20^oC. Why are these proteins shipped with ice-packs instead of with dry-ice? Also, wouldn't be even better if I were to store the proteins at -80^oC?

ANSWER #2) Glycerol/water (50% v/v) will remain in an aqueous state at -20^oC, but will begin a phase transition as it approaches -30^oC. To avoid a phase transition which may be damaging to the protein, the samples should not be stored at temperatures much below -20^oC. We ship our proteins on ice-packs which are originally chilled to -20^oC, and which will maintain a temperature of at least 4^oC or colder for the duration of the shipping process. The brief exposure to elevated temperatures during the shipping process is not detrimental to the protein.

QUESTION #3) The glycerol/water formulation is viscous and is sometimes difficult to pipette. Do you have any suggestions on how to handle material that is formulated in glycerol/water?

ANSWER #3) When preparing to pipette the stock glycerol/water solution, remove the sample from -20^oC storage and place it on ice for 10 to 15 minutes. The sample will become less viscous and thus easier to pipette. In addition, do not immerse the pipette tip any further than necessary to retrieve the sample, and you will avoid having material adhere to the outside of the pipette tip.

QUESTION #4) I have a special application for a protein in which I cannot tolerate even trace amounts of glycerol or free amines. Will HTI reformulate the protein to a buffer of my choice?

ANSWER #4) Yes, we will reformulate any protein to a buffer of your choice. Occasionally we may have to point out that a particular buffer is not good choice and we would therefore try to suggest alternatives. Reformulation will be done for an additional charge of $250 unless the order for that product is $2,500 or more. At that level the reformulation will be performed free of charge.

QUESTION #5) We have purchased your bovine factor Xa and thrombin with the intention of using it to cleave our fusion protein. What concentration of factor Xa and thrombin should I use for this purpose?

ANSWER #5) The first thing to realize is that factor Xa or thrombin may exhibit different kinetic behavior when used to process different fusion proteins. Therefore a set of conditions which are known to work well with one fusion protein may not be adequate for another. We suggest starting with an enzyme to substrate molar ratio of 1:50, in a buffer adjusted to physiologic pH and ionic strength (such as 20mM Hepes, 0.15M NaCl, pH 7.4). Generally this ratio of enzyme to substrate will be sufficient, but experimenting with different conditions or perhaps raising the pH slightly to 7.8 may be necessary to optimize cleavage conditions. It is important to note that although these enzymes are relatively specific for defined cleavage sites, situations can arise where other sites in the fusion protein are cleaved. After all the P and P' sequences of various cleavage sites found in the natural substrates for these enzymes are known to be different. By adjusting enzyme to substrate ratios and by experimenting with different buffer systems, it may be possible to gain specificity.

Back to top