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CIM Antibody Core
F.A.Q.
Frequently Asked Questions

Questions about...

Immunization Technology

What is current turnaround time for a custom antibody?
About three months.


What is your success rate of antibody production?
Our overall success rate is >90 %.


What antibody is qualified as a hard case and how would it be treated differently from the normal order?
Any antibody that failed or was of poor quality generated using conventional immunization techniques. Our willingness to try difficult antibodies is based our confidence in our genetic immunization technology which proves to have advantage over conventional methods for preserving natural protein folding and offering purer products. We are also successful in generating antibodies to mouse proteins in mice.


What animals do you use to produce antibodies?
Two outbred CD1mice are used routinely for each antigen. Please contact us if you would want other animals to be used.


What should I use for the secondary antibody?
For the secondary antibody, you should use an anti-mouse IgG.


How should I store sera?
All sera have been prepared in 50% glycerol. We suggest that sera be stored at -20°C.


What is your protocol for Western blotting?

Western Blotting Protocol

1. Add SDS loading buffer to a crude E. coli extract containing 50 - 500 ng of GST/Ag fusion protein and heat at 5 minutes 97 oC. Spin the sample 5 min at the full speed in a microcentrifuge , and load immediately onto a pre-made 4 - 20% gradient gel (Express). Run gel in 1 X HEPES buffer.

2. Transfer protein onto a Shleicher & Schuell Nitrocellulose membrane (0.4 u) 1 X Transfer Buffer with a BioRad Semi-Dry Transferring Cell 1 h at 12 V.

3. Block in 1X TBST containing 5% milk for 1 h at RT or ON at 4oC with agitation. .

4. Add anti-serum to a proper amount of 1 X TBST containing 5% milk (enough to cover the membrane) at 1:2000 or 1:1000 dilution. Shake the blot 1 to 2 h at RT.

5. Wash the blot with 1X TBST 3 times. Every time 5 minutes at RT.

6. Incubate the blot with PIERCE Immuno Pure Rabbit Anti-mouse IgG + IgM (H + L), HRP Conjugated, at a 1:5000 dilution for 1 h at RT.

7. Wash 3 times as step 5.

8. Incubate the blot with SuperSignal West Pico Chemiluminescent Substrate (Product # 34080) as described by the manufacturer.


Buffers:

10 X HEPES Running Buffer (1L):
    121g Tris
    238g Hepes
    Dissolve in 1 L H2O
    Add 10 g SDS

10 X TBST (10 L):
    121 g Tris, dissolved in 4 L H2O
    Adjust pH to 8.0
    Add 800 g NaCl
    Add 100 ml Tween-20
    Fill H2O to 10 L

10 X Transfer Buffer (1L):
    30.3 g Tris
    144 g glycine
    Dissolve in 1 L H2O. pH should be 8.3 (do not adjust)

To make 1 X, add Methanol to 10%



Antigen/Targeting Design

What are AI scores and how are target regions chosen?
The antigenecity index (AI) that is used for locating target sequence for a given antigen is primarily based on the Jameson and Wolf algorisms. Customer may specify a region of interest. If this region is a small peptide (20 aa or smaller), it generally will be used directly. If customer suggests a larger region, unless specifies that this entire region must be used, this segment will be subjected to an analysis and the sequence with the best AI score will be used, whether it is a peptide or a segment (normally within 100 aa).

Assays

Can I use [a particular antibody] with an immunohistochemical assay?
The immunohistochemical assay can be difficult to perform. We have had many researchers report success using our antibodies with an immunohistochemical assay. However, others have not had success. We do not perform this assay internally, and so it is impossible to state whether this assay will work for a particular antibody. You could order some and try it out for yourself. Beyond that, you could write a brief question that we could forward to other researchers who have used this antibody. You would not receive any information about who else used this, but they would receive your contact information, and they could write back to you if they choose to.


I noticed that on your website it says you used an anti-mouse IgG + IgM. Does the anti IgM make a difference in the western blotting? And if so, where can I obtain this secondary antibody?
Yes, the anti IgM DOES make a difference. The secondary antibody we use is from Pierce, product number 31457: ImmunoPure Rabbit Anti-Mouse IgG+IgM, (H+L), Peroxidase Conjugated.

Antibody Ordering

How do I order a custom antibody?
To order a custom antibody, please use an online custom antibody form at our website (
http://www.innovationsinmedicine.org/antibody/form.html). Enter all the information and note how you would like to have regions chosen if you have preferences. As soon as you submit the form online, we will process the order. Note that we are not currently beginning work on new custom antibody orders, but we plan to begin doing so again within the next few months.


How do I order a pre-made antibody?
To place a pre-made antibody order, you must fill out the online order form at:
http://www.innovationsinmedicine.org/antibody/formpremade.html


How much serum can I order?
For the initial production, we normally use two mice from which approximately 2 mls of serum can be produced. If it is a custom antibody, we would want to guarantee that the primary requester gets at least 500 ul serum. For the remaining serum, we would like to provide to as many researchers as possible. We normally send out 100 ul for all researchers. If more is needed, please contact us.



How do I know if my custom antibody is ready and how to get it?
We will contact you by email as soon as a positive result is obtained in our test. One other way to find out is to check the status online. When it is ready, a Western image will be posted. Please follow "How to order pre-made antibodies" for ordering and shipping.

Pricing

How much do the antibodies cost?
Detailed pricing information can be found on the CIM Antibody Core home page:
http://www.innovationsinmedicine.org/antibody


How does the cost recovery program affect my order if it was placed before the program was instituted?
If you have ordered a custom antibody before Sept 28, 2003, you will be offered 100 ul of serum free. Additional aliquots will be charged according to the prices listed on our website.

Shipping

How are antibodies shipped?
We ship through FedEx and Airborne (DHL). When you order your antibodies, you will specify which of these shippers to use, and provide a shipping account number.


Billing

How is billing handled?
When you place your order for antibodies to be shipped to you, we require a Purchase Order Number (PO#). We do not provide PO#s. Such numbers should meet the requirements of your institution. The order form also requests billing contact information, and a billing address (if different from the shipping address).

After antibodies have been shipped to you, a bill will be mailed to the billing address (or shipping address if a separate billing address is not provided). The Purchase Order Number you specified and all details of your order will be included on the invoice. Your institution will remit payment via check or money order. (Arizona State University researchers may use IDR.)


What is the mailing address for the Antibody Core?
Our mailing address is:
Arizona State University
Center for Innovations in Medicine
c/o Pattie Madjidi
P.O. Box 875901
Tempe, AZ 85287-5901



What is your contact Number
Jerry Lin
(480) 727-9582


What is your Federal Tax ID #?
Our Federal Tax ID # is: 860196696


Who do I write checks out to?
When you receive your shipment and invoice, you will make checks payable to:
Arizona State University


Still have questions that were not answered on this page?
    
Contact us



Web page created 16 August 2001. Last modified 5 July 2006.