Anti-AMHR2 antibody - Aminoterminal end (ab75845)

Thanks for your kindly reply , after I contacted with this customer she would like to have a credit note,
could you please help this customer to create a credit note?
Thanks for your kindly assistance.
Best regards

ANSWER:

Thank you for contacting us.

Your credit note ID is XXXXXX

I am sorry that this antibody did not perform as stated on the datasheet, I have asked our Finance department to issue a credit note for you. The credit note may be used in one of the following ways:

(1) Redeemed against the original invoice if this hasn't already been paid.
(2) Held on the account for use against a future order.
(3) A full refund can be offered where no other invoices are outstanding.

Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website.

The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

Thanks for your kindly reply, and sorry about the attached error in last letter, I attached the results from ab75845 and ab64762 in this letter again, and I also recheck the marker with this customer, please check this and offer any suggestion to this customer.
Thanks for your kindly help
Best regards

ANSWER:

Thanks you for your email. Dr. Tanya is away this week so I am dealing with her inquiries.
I am sorry that the product did not perform well. I can provide a free of charge replacement, refund or credit note. Could you please confirm how the customer would like to proceed?

Dear technical support team:

This customer has purchased ab75845 (Anti-AMHR2 antibody - Aminoterminal end) and has conducted the wb several times with human sample. The results show wrong band size and more bands, therefore this customer wants to ask for your help to modify her experiment step, could you please offer any suggestion to improve her results? I attached the image in this letter and his experiment step as follow:

1. Order details:


Batch number: GR61389-1

Abcam product code: ab75845

Antibody storage conditions (temperature/reconstitution etc) : -20℃




2. Please describe the problem: wrong band size, and more bands!



3. On what material are you testing the antibody in WB?

· Species: Human

· What’s cell line or tissue : HCC cell line, Hep3B and ML

· Cell extract or Nuclear extract: Cell extract

· Purified protein or Recombinant protein: No



3. The lysate


How much protein was loaded: 15 ug

What lysis buffer was used: RIPA



What protease inhibitors were used: Na3VO4, EDTA, PMSF, NaF

What loading buffer was used: 4X loading buffer(with DTT)

Phosphatase inhibitors : Na3VO4, NaF

Did you heat the samples: temperature and time: 95℃,5 mins




4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: reducing gel

Reducing agent: DTT

Gel percentage : 10%

Transfer conditions: (Type of membrane, Protein transfer verified): PVDF membrane, 5% methanol 1X Transfer buffer.




5. Blocking conditions


Buffer: 5% BSA(TBST)

Blocking agent: BSA

Incubation time: O/N

Incubation temperature:4 ℃




6. Primary Antibody


Species: Rabbit

Reacts against: Reacts with Human


· At what dilution(s) have you tested this antibody: 1:50

· What dilution buffer was used: 5% BSA(TBST)

· Incubation time: 2 hr

· Incubation temperature: RT (25℃)

· What washing steps were done: TBST wash three times,10 min each time



7. Secondary Antibody


Species: Gt X Rb IgG(H+L) HRP , 4℃, Millipore

Reacts against: Reacts with Rabbit

At what dilution(s) have you tested this antibody: 1/2000,5% milk

Incubation time: 50 mins

Wash steps: TBST wash three times,10 min each time

Fluorochrome or enzyme conjugate: enzyme conjugate

Do you know whether the problems you are experiencing come from the secondary? I sure it’s no problems.




8. Detection method
ECl, ECl+, other detection method:
Western HRP Substrate.WBKLS0500 - Millipore

9. Did you apply positive and negative controls along with the samples? Please specify. No

10. Optimization attempts

· How many times have you tried the Western? 3 times

· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): No

· Do you obtain the same results every time e.g. are background bands always in the same place? Yes, the same results.

· What steps have you altered? Concentration of primary antibody



Could you please help this customer to solve the problem?

Thanks for your kindly help

Best regards

ANSWER:

Thank you for your enquiry regarding ab75845 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

1) Transfection:

a) Could you please explain what these abbreviations represent provided on the Western blot image?

ML shControl,

ML shAMHR2 (G9),

ML shAMHR2 (B10),

3B transfection control,

3B siAMHR2 (s5),

3B siAMHR2 (s6)

b) Looking at the signal of the loading control it seems that not the same amount of protein was loaded onto the gel. Could you explain if the customer has performed any time-course experiments which may influence the expression of the actin. What is the sizeof the actin on the blot, could you please label the image with MW for the actin as well?

c) Does the construct contain full sequence or fragment of the target protein? Does it correspond to human AMHR2 sequences?

c) Could you please confirm if the target protein is tagged, if so which tag(s) was used?

c) How was the efficiency of the transfection checked?

2) Secondary antibody:

a) Does the detection system work fine? Have you used it successfully with another primary antibody?

b) Have you run a no primary - only secondary antibody - control to see if any of the non-specific bands are due to the secondary or not? If you have not done yet, I would advise you to check it.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.