Anti-KDM1 / LSD1 antibody - ChIP Grade (ab17721)

I work on Toxoplasma Histone Demethylases and currntly I am focusing on the Toxoplasma LSD family. I have order the chip grade rabbit polyclonal ab (ab17721) and cross react perfectly with the Toxoplasma LSD1 in stainings as well as in WB. I want to perform more detailed localization studies and see if Toxoplasma LSD1 co-localizes with other molecules inside the nucleus and especially the centromeres. For this I would like to use a mouse a-LSD1 ab. I have search your products and I found that you have 3 anti mouse a-LSD1 abs (ab-909966, ab-31954, ab-6935) . I would like to ask you which is the exact region of the human recombinant ag that you used in order to produce the ab-909966? Secondly I would like to ask if I could have an aliquot of these abs so I could test them against the Toxoplasma LSD1 and see if they cross-react before I order any of them .

ANSWER:

Thank you for your enquiry.

We do not have small sample sizes available. In lieu of this, we do have a testing program which you may be intersted in. I have described it below using ab90966 as an example. But the promotion could be applied to any of the antibodies you have mentioned here. Please let me know if you would like to take advantage of the promotion with 1 or more antibodies or if you have any questions.

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Thank you very much for your interest in ab90966.

To our knowledge, ab90966 has not been tested in Toxoplasma. Therefore, I can offer a discount off a future purchase if you buy ab90966 now, test it in Toxoplasma and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of: 1 free primary antibody. If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab90966 in Toxoplasma Iwill then send a discount code. This code must be issued before purchasing ab90966 so please wait for my reply before ordering.

2. Purchase ab90966 either by phone, fax, or online (www.abcam.com).

3. Test it in Toxoplasma.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: http://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for anyprimary antibodyordered and the discount code is valid for 4 months after issue.
Please remember that submission of the Abreview is sufficient for the discount code to become active.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab90966 turns out to be unsuitable forToxoplasma you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

Dear xxxx

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 he would like to have a credit note,

could you please help her to create a credit note?


Thanks for your kindly help
Best regards

ANSWER:

I am sorry that the protocol suggestions made have not lead to a significant improvement in the non-specificity observed. It does however appear that the non-specificity has been reduced. I would suggest to your customer that it would be worthile trying to optimise the protocol further, for example using a higher dilution of the antibody (1/2000) as well as reducing the amount of protein loaded on the gel (to 10 ug/well). This may lead to further improvements in the results observed.

I can however understand the frustrations of your customer and the time and effort they ahve put into optimising the results with this antibody. I ahve therefore generated a credit note as requested. The credit note ID is xxxxx.

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

Dear technical support team:

This customer has purchased ab17721 (Anti-KDM1 / LSD1 antibody - ChIP Grade) 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: GR50119-1
Po:
Abcam product code: ab17721
Antibody storage conditions (temperature/reconstitution etc) -20oC
2. Please describe the problem (high background, wrong band size, more bands, no band etc). More bands, no specific band
3. On what material are you testing the antibody in WB?
· Species: Human
· What’s cell line or tissue: K562, HL60, U937, and Jurkat…..
· Cell extract or Nuclear extract: Cell extract
· Purified protein or Recombinant protein: purified
3. The lysate
How much protein was loaded: 20-30 µg
What lysis buffer was used: RIPA
What protease inhibitors were used: inhibitor cocktails
What loading buffer was used: 4X sample buffer (1M Tris-HCl pH 6.8, SDS, Glycerol, BPB)
Phosphatase inhibitors : sodium fluride, sodium orthovanadate
Did you heat the samples: temperature and time: 95oC 3-5 mins
4. Electrophoresis/Gel conditions/ Transfer conditions
Reducing or non reducing gel: Reducing
Reducing agent: BME
Gel percentage : 8% SDS PAGE
Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, pre-stained marker
5. Blocking conditions
Buffer: TBST
Blocking agent: 5% milk
Incubation time: 1-2h
Incubation temperature: RT
6. Primary Antibody
Species:HumanReacts against: KDM1/LSD1
· At what dilution(s) have you tested this antibody:1/1000
· What dilution buffer was used: TBST in 5% Milk
· Incubation time: O/N
· Incubation temperature: 4oC
· What washing steps were done: more than three times using TBST buffer
7. Secondary Antibody
Species:Reacts against: Rabbit IgGAt what dilution(s) have you tested this antibody: 1/5000-1/10000Incubation time: 45-60 minutes
Wash steps: 3 times, 10 minutes each time, TBST buffer
Fluorochrome or enzyme conjugate: enzyme conjugate
Do you know whether the problems you are experiencing come from the secondary? No
8. Detection method
ECl, ECl+, other 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? 3 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? Higher dilution of 2Ab
Could you please help this customer to solve the problem?
Thanks for your kindly help
Best regards

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab17721. This antibody has been used in house with 5% BSA used as the blocking agent. I would therefore suggest that you try thins blocking agent, in TBST to block the membrane, as well as in the diluent of the primary and secondary antibody. This change from milk blocking to BSA blocking can sometimes have a dramatic effect on the specificity and strength of signal observed with an antibody (as is illustrated on the datasheet of http://www.abcam.com/ab9385). I would also suggest increasing the concentration of your SDS-page gel, from 8 % to 12 % to improve the resolution of the bands.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Thank you for sending the replacement antibody (Lot: GR79241-1). I have repeated the ChIP experiments in either control or LSD1 KD cellsusing: our original LSD1 antibody that was working (959450), the antibody that did not work for us (GR50119-1) and the replacement antibody (GR79241-1). As you can see from the results below, the new replacement antibody seems to work fine, similar to our original antibody. Again, the antibody which we had trouble with did not perform for ChIP experiments.

As this replacement antibody seems suitable for ChIP, I was wondering how many vials you have of this specific lot in stock as I would like to purchase a number of vials? For the optimisation of this antibody, we have carried out a lot of work and timein testing these antibodies. This includes lentivirally infecting cellsto knockdown LSD1, sorting the infected cells, performing ChIP using other control antibodies (both positive and negative) as well as using different lots of LSD1 antibody and qPCR for analysis. We've had to do this twice now (once with the original lot that didn't work and again to compare the new lot to the other antibodies), which has cost us a significant amount of money. Would it be possible to receive a discount for a bulk order of these antibodies, taking into account a bulk order purchase and the time and money spent testing the different antibodies?



Kind Regards,

ANSWER:

Thank you for your kind feedback. I am glad to hear that the antibody finally works for you. I sincerely apologize for the inconvenience caused due to failing of lot GR50119-1.

We acknowledge your request and you being a valued customer of Abcam, we can consider providing discount. We have 28 vials of GR79241-1 in stock, could you tell me how many vials you would like to order?



I will look forward to hearing from you soon.

The order number is UTZ930445. Please can you confirm a delivery date?

Regards,

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1061279.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

PS: You will receiving the product tommorrow morning.