FAQs - Frequently Asked Questions

BD Gentest™ Antibody Products
Below are the answers to many frequently asked questions submitted by our customers. If the following does not address your questions please contact info_gentest@bd.com.

What is included in an antibody kit?

How many experiments can I perform with one immunoinhibition kit?

How many experiments can I perform with one Western blotting kit ?

Can I order the primary antisera separately from the other kit components for a discounted price?

For a Western blot, what other materials do I need to purchase?

What products do you recommend?

What is the predicted molecular weight (estimated migration on SDS-PAGE) of this protein?

How specific is this antisera?

Why do the DAIICHI inhibitory antibody kits contain normal immune serum, whereas the BD Gentest inhibitory antibody kits contain Tris buffer?

Do you sell control sera separately?

Q: What is included in an antibody kit?
A: We provide antibody kits for Western blotting (immunoblotting) or inhibition. These kits are manufactured by BD Biosciences. DAIICHI PURE CHEMICALS CO., LTD. also provides single vials of antiserum for use in Western blotting and inhibition.

• BD Gentest™ Inhibiton kit
  A BD Gentest Inhibition kit contains 0.4 ml primary antisera and 1 ml 25 mM Tris pH 7.5 (for use as a diluent).
• BD Gentest™ Western blotting kit
  Each BD Gentest Western blotting kit contains enough primary and secondary antisera (0.4 ml or more) to perform at least 20 mini blots (200 ml working reagent). Qualitative positive (and negative) controls are sold separately.
  

Q: How many experiments can I perform with one immunoinhibition kit?
A: The number of experiments that can be performed is dependent on the inhibitory antibody used and the sensitivity of the detection method. DAIICHI inhibitory antibodies contain 0.2 ml antisera. BD Gentest kits contain 0.4 ml antisera.

DAIICHI Inhibition Antisera

* for maximum inhibition

BD Gentest Inhibition Kits

Q: How many experiments can I perform with one Western blotting kit?
A: Each Western blotting kit contains enough primary antisera (0.2 ml or more) to perform at least 20 mini blots (200 ml working reagent). In many cases twice as many blots can be performed when very sensitive developing techniques (enhanced chemiluminescence) are used. Details can be found on individual data sheets.

Q: Can I order the primary antisera separately from the other kit components for a discounted price?
A: The primary antisera is not sold separately from the other kit components because the essential cost of the kit comes from the development and characterization of that primary antisera.

Q: For a Western blot, what other materials do I need to purchase?
A: For Western blotting kits and DAIICHI vials of antiserum, you will need to purchase a qualitative positive control, developing agent, and buffer components separately.
Note: The buffer components necessary for blocking, washing, and antibody dilution are dependent on the antibody used. Exact blotting conditions for each antibody are defined on their product data sheets. You must consult those sheets for appropriate buffers and blotting conditions.

Q: What products do you recommend?
A: Our recommendations are shown below.

• Qualitative positive control
  A qualitative positive control is an important tool for judging the relative expression of a protein and for ascertaining its identity (mobility on gel). We recommend using our microsomes containing lymphoblast- or baculovirus-expressed (BD Supersomes™) enzymes for use as positive controls. In both cases, control microsomes (containing no cDNA-expressed enzyme) can be used as negative controls.
• Secondary Antibody
  Our primary antibodies were developed in mouse, rabbit, or goat. The appropriate secondary antibody will bind the primary antibody and be conjugated to the enzyme of your choice. We use alkaline phosphatase conjugated antibodies for colorimetric detection and peroxidase conjugated antibodies for enhanced chemiluminescent (most sensitive) detection on Western blots.
  
  

  Description	*Catalog No.	Dilution	Dilution buffer
  Alkaline Phosphatase conjugated Anti-Goat lgG	A2168	1:5,000	0.5% milk/TBS
  Alkaline Phosphatase conjugated Anti-Mouse lgG	A2418	1:5,000	0.5% milk/TBS
  Alkaline Phosphatase conjugated Anti-Rabbit lgG	A0418	1:5,000	0.5% milk/TBS
  Peroxidase conjugated Anti-Goat lgG (whole molecule)	A5420	1:2,000	0.5% milk/TBS
  Peroxidase conjugated Anti-Mouse lgG (whole molecule)	A9917	1:2,000	0.5% milk/TBS
  Peroxidase conjugated Anti-Rabbit lgG (whole molecule)	A6154	1:2,000	0.5% milk/TBS

  *Reagents purchased from Sigma.

• Developing Agent
  Developing agents are not included in BD Gentest or DAIICHI manufactured kits. they may be obtained from Kirkegaard & Perry Laboratories (KPL), Gaithersburg, Maryland USA, www.kpl.com, 301-948-7755 or Pierce, Rockford, Illinois USA, www.piercenet.com, 815-968-8148.
  
  Colorimetric Detection:
  BCIP/NBT Phosphatase Substrate (1 Component)
  KPL Cat. No. 50-81-07
  
  Enhanced Chemiluminescent Detection:
  LumiGLO Chemiluminescent Substrate kit
  KPL Cat. No. 54-61-00
  
  Supersignal West Pico Chemiluminescent Substrate
  Pierce Cat. No. 34080ZZ
  
  
• Blocking buffer/Antibody Dilution buffer/wash buffer components
  Some buffer components you may need are shown below. Consult data sheet for exact components required for antibody of interest.
  
  

  Description	Company	Catalog No.
  Carnation Nonfat Dry Milk	grocery store	*N.A.
  Bovine Serum Albumin	Sigma	A7888
  Polyoxyethylenemonolauratesorbitan (Tween-20)	Sigma	P1379
  Polyvinyl Alcohol (PVA) average m.w. 30,000-70,000	Sigma	P8136

  *Not Applicable
  

Q: What is the predicted molecular weight (estimated migration on SDS-PAGE) of this protein?
A: The following molecular weight designations have been predicted based on the amino acid sequence of each enzyme. The apparent molecular weight, as deduced from mobility on an SDS PAGE, may differ from the predicted molecular weight. When an apparent mobility is known to differ from the predicted molecular weight, it is noted in the table.

Predicted Molecular Weight

¹Human CYP3A4 (57.3 KD) migrates more quickly (further) than human CYP3A5 (57.1 KD) on an SDS PAGE even though their predicted molecular weight indicates otherwise.

Q: How specific is this antisera?
A: Antibody specificity tables are available in Technical Resources.
Human P450 Inhibitory Antibodies
Human P450 Immunoblotting Antibodies
Human UGT Immunoblotting Antibodies
Human FMO Immunoblotting Antibodies
Rat P450 Inhibitory Antibodies
Rat P450 Immunoblotting Antibodies
Dog P450 ELISA Antibodies

Q: What is the difference between DAIICHI inhibitory antisera and BD Gentest inhibitory antibody kits?
A: Currently, the principal difference between the DAIICHI inhibitory antibodies and the BD Gentest inhibitory antibodies is that DAIICHI are whole serum-based polyclonal antisera, whereas BD Gentest are ascites-based monoclonal antibodies. The total protein concentration of the polyclonal antisera is 80 to 100 mg/ml. About 0.1 to 2.5% of these serum proteins represent specific antibody. Therefore, the use of normal or nonimmune serum as a diluent or negative control is recommended to ensure that any effect seen is not just a nonspecific protein binding effect.

All of our monoclonal antibodies are standardized with 25 mM Tris pH 7.5 to a protein concentration of 10 mg/ml (from about 30 mg/ml initially). About 10 to 50% of the total protein in ascites is immunoglobulin with 90% being specific antibody. Therefore we would use Tris buffer or a nonspecific ascites preparation as a negative control.

Q: Do you sell control sera separately?
A: We do not sell control sera separately. An unrelated antisera or normal sera from the same animal can be used as a negative control. Normal sera can be obtained from a vendor such as Jackson ImmunoResearch Laboratories (610-869-4024; cuserjaxn@aol.com; West Grove, PA USA).