Anti-IKB alpha (phospho S32 + S36) antibody (ab47752)

Thanks for your kindly reply, after contacting with this customer and passed these suggestions to him then he modified the experiment step as your suggestions,

but it did not improve the result. I attached the result in this letter, please check it and she would like to have a credit note,

could you please help him to create a credit note?


Thanks for your kindly help
Best regards

ANSWER:

Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note. This can then be redeemed against the invoice of a future order.

Credit ID: #####

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

Dear technical support team:

This customer has purchased ab47752 (Anti-IKB alpha (phospho S32 + S36) antibody) and has conducted the wb several times with mouse sample. The results show no major band and high background, therefore, she wants to ask for your help to modify her experiment step, could you please offer any suggestion to help her?

I also attached her image in this letter and her experiment step as follow:


1. Order details:

Batch number: gr72085-1

Abcam product code: ab47752

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


2. Please describe the problem (high background, wrong band size, more bands, no band etc).

No band and high background

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

Species: Mouse

Cell line : RAW264.7

Cell extract or Nuclear extract: Cell extract

Purified protein or Recombinant protein:



3. The lysate


How much protein was loaded: 50ug/well

What lysis buffer was used: RIPA buffer (Gene Star)



What protease inhibitors were used: no

What loading buffer was used: abcam loading buffer

Phosphatase inhibitors: Millipore

Did you heat the samples: 95℃, 5 min




4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: Reducing

Reducing agent:

Gel percentage : 10%

Transfer conditions: PVDF (millipore), 110V, 70 min




5. Blocking conditions


Buffer: PBST(0.1% Tween-20)

Blocking agent: milk

Incubation time: 2 hrs

Incubation temperature: 4℃




6. Primary Antibody


Species: Rabbit polyclone

Reacts against:


· At what dilution(s) have you tested this antibody: 1:500

· What dilution buffer was used: PBST(0.1% Tween-20)

· Incubation time: overnight

· Incubation temperature: 4℃

· What washing steps were done: wash 10 min at three times



7. Secondary Antibody


Species:

Reacts against: Rabbit IgG (HRP)

At what dilution(s) have you tested this antibody: 1:5000

Incubation time: 2 hrs

Wash steps: wash 10 min at three times

Fluorochrome or enzyme conjugate: enzyme conjugate

Do you know whether the problems you are experiencing come from the secondary?


This secondary antibody was used on same species of primary antibody (ab32581, IkB), that band was very clear.

8. Detection method
ECl, ECl+, exposure time for 10 min



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? Two times

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

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

What steps have you altered? Protein loading and concentration primary antibody

Could you please help this customer to solve the problem?

Thanks for your kindly help

Best regards

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will provide us with vital information for our monitoring of product quality. I would like to reassure you that this antibody is tested and covered by our guarantee forWB and mouse.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results. I can suggest youmay possibly haveregrettably received a bad vial.

I can recommend you may like to consider trying the following options:

1. Could you confirm thequality of the sample has been assessed using a loading control?

2. I can recommend to try blocking in BSA rather than milk. Changing blocking agent can sometimes help optimize the results. There is one Abreview published on the datasheet from a customer who mentions that results were better with BSA.

Alternatively, or if these tips do not work, I am pleased to offer you a free of charge replacement or credit note in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

the shipping adress is correct! Thank you very much and best wishes,

ANSWER:

Thank you for your reply and confirming the shipping address. I just like to double check the address before I send the replacement. I have processed your request to receive ab59196 as a replacement for ab47752. Your new order number is *******.

, sorry for the late reply. I have the details about the original order of ab47752 for you: It was ordered on 03-15-2011 by . The order number, which I could find on our delivery invoice from the central hub of the was , if that helps you, although I am not sure if this is only an internal number. I agree that the time span from the ordering date to the date of this communication exceeds your 6 months policy, but naturally I used the antibody right away after it arrived. I hope this delay will not be a problem. Thank you and best regards,

ANSWER:

Thank you for your reply,

I believe I have found your original order and I would be happy to send you ab59196 as a replacement. I would just like to confirm the shipping address before I process the replacement. The last order was shipped to:

If this is correct please let me know and I will have the replacement antibody shipped to y

yes, I do have bands basically everywhere, both below and above my band of interest. I would rule out degradation of my protein anyways, because probing for IkappaB with a non-phospho-specific antibody I do only get one band. Anyways, you offering me a replacement or refund is highly welcome! To tell the truth: that really distiguishes your company from the competition! I would probably opt for the replacement antibody. Is it possible to get a different antibody than the discussed one? That would be great since I doubt that another vial of anti-phospho IkappaB would be much helpful, and since we are frequently purchasing the anti-phospho JNK (ab59196) from you, so that one would be more useful for me. Please tell me how we can handle this. Thank you very much and best regards,

ANSWER:

Under our Abpromise this antibody is guaranteed for six months to work in western blot in human, mouse, rat and cow samples. Since it is not working in your hands, then I would be happy to send you a replacement. If you wish to receive ab59196 as the replacement then that is not a problem. To be able to send you the replacement antibody could you provide me with the original order details please? Even the date it was ordered or who actually place the order would be helpful. Once I get this information, I will be able to send you the replacement vial.