Anti-iNOS antibody (ab15323)

Thanks for your kindly reply, after contact this customer, she would like to have a credit note,

could you please help her to create a credit note ?

I deeply appreciate your great assistance!

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: 20559

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.

Thanks for your kindly reply, after contacting with this customer and passed these suggestions to her then she 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 help this customer to solve this problem.

Thanks for your kindly help
Best regards

ANSWER:

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or credit note in compensation.

I look forward to hearing from you with details of how you would like to proceed.

Dear technical support team:

This customer has purchased ab15323 (Anti-iNOS antibody) and has conducted the wb several times with mouse sample. The results show no band, 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: gr67797-1

Abcam product code: ab15323

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


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

No band

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

Species: mouse

What cell line or tissue :aorta

Cell extract or Nuclear extract: cell extract

Purified protein or Recombinant protein:

3. The lysate

How much protein was loaded: 30micro gram

What lysis buffer was used: np40

What protease inhibitors were used: Roche cocktail

What loading buffer was used: 6x sample dye

Phosphatase inhibitors Na3VO4

Did you heat the samples: temperature and time: 95/5 min

4. Electrophoresis/Gel conditions/ Transfer conditions

Reducing or non reducing gel: non

Reducing agent:

Gel percentage : 10and 15

Transfer conditions: (Type of membrane, Protein transfer verified): 100v 1hr




5. Blocking conditions

Buffer : TBS

Blocking agent: 5%milk:

Incubation time: 1hr

Incubation temperature: RT

6. Primary Antibody

Species: R

Reacts against:


At what dilution(s) have you tested this antibody: 1:500 or 1:1000

What dilution buffer was used: 5% milk /TBS

Incubation time: 4℃ on

Incubation temperature:

What washing steps were done:

7. Secondary Antibody

Species:

Reacts against: Rabbit

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

Incubation time: 1hr

Wash steps: 10min with 1% TBST for 3 times

Fluorochrome or enzyme conjugate:

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

8. Detection method
ECl,

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? 2 times

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

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

What steps have you altered? Prolong the wash step

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 enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab15323 is tested and covered by our 6 month guarantee for use inWB andmouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimize the results from ab15323. I would also appreciate if you can confirm some further details:

1. I can suggest to run the samples on a reducing gel to ensure the sample runs at the correct molecular weight and that it is in the correct conformation for detection by the antibody.

2. I can recommend to try BSA rather than milk to block. Changing blocking agent can sometimes help to improve the results.

3. Could you confirm if thetransfer to the membraneand quality of the sample have been assessedusing a loading control?

4. Could you confirm if the current vial of secondary antibody is working well with other primary antibodies? The concentration may need to be Increased.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Thank you very much.



The customer prefers to receiveab49999 as a replacement.



Please confirm.




Warm Regards,

ANSWER:

Thank you for your note.
This is to let you know that I have confirmed and agreed on placing a new order for your customer - for one vial of ab49999 (exchange for the original product ab15323) as a free of charge replacement. I hope the second vial will work as it is expected, and please do let me know how you are getting on with this product.
Dear Colleague,
Please add 1 vial of ab49999 to the next shipment for Zotal.
Original CCE ID: 3410854
Original Order: 1003928
Problem is: Non-specific bands in Western blot application
Many thanks.

Please see below the details of customer's complaint. Attached is the image. Thanks in advance for your reply. Antibody code: AB15323 Batch number: GR592361 Antibody storage conditions (temperature/reconstitution etc) -20, after reconstitution 4C Description of the problem (high background, wrong band size, more bands, no band etc.) Many bands detected. Specifically around 130kD Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) Mouse brain homogenate Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) Buffer (Tris, glucose, glycerin, MgCl2, EDTA) Protease inhibitor , Na3VO4, PMSF, NaF. Heating 96C 2-3 min. Amount of protein loaded 50mcg Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) 9% with SDS Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) Buffer transfer: Tris 6g, Glycerine 28.8g, Ethanol 400mL DDW ad 2L Block 5% skim milk 1-2hr RT Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Abcam AB15323, in 5% BSA with sod. Azide. dilution 2mcg/mL (1:200) incubation 16hrs 4C. 3 washes TBS-T 10 minutes each Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Jackson, 1:10,000 anti rabbit in 5% skim milk, 1hr followed by 3 washes 15min each Detection method (ECL, ECLPlus etc.) ECl Positive and negative controls used (please specify) Non. another Ab worked using the same samples. Optimization attempts (problem solving) How many times have you tried the Western? 2 times at slightly different conditions Have you run a "No Primary" control? no Do you obtain the same results every time? e.g. are the background bands always in the same place? Yes What steps have you altered? longer blocking time Additional Notes Another antibody from a different company (borrowed from other lab) worked using the same samples.

ANSWER:

Thank you for your enquiry regarding ab15323 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody. I can confirm that ab15323 detects the inducible nitric oxide synthase (iNOS) in mouse, human and rat and it is suitable for Western blot application. Looking at the image, it seems that the non-specific bands are at the lower range which may imply that they represent some sort of degradation products. 1) Could you confirm if the mouse brain samples are normal or the tissues/animals were treated with some drugs to induce iNOS? 2) It would help me if you could confirm if the alternative antibody recognizes the same target or different proteins? Would you mind sharing with me the different results of this antibody and the alternative product so that I could take a look at the different signal? Would your customer prefer to get a new vial as a free of charge replacement or a credit note? I look forward to hearing from you soon.