Anti-Dnmt3a antibody (ab4897)

Thank you for your phone call.
I am sorry that the antibody is not working as expected. As we discussed, I have arranged for a free of charge replacement with ab4897. Please see details below. The item is currently backordered, but the estimated delivery date is set for xxx. I Hope that is OK.

Also, below please find the discount code for trying ab4897 in mouse and rat and additional information on how it works.

DISCOUNT CODE: xxx
Expiration date: xxx
Value: xxx

I am very pleased to hear you would like to accept our offer and test ab4897 in mouse and rat. This code will give you xxx off your next order before the expiration date. To redeem this offer, please submit an Abreview for mouse and/or rat and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/abtrial.

Here is the information regarding your free of charge replacement:

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 xxx with ab4897.

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.

Thank you very much for your interest in ab4897.

To our knowledge, ab4897 has not been tested in Mouse and Rat.It is very likely that ab4897works in these species since thehomolgy between the immunogen peptide and the proteins of interest is 100% (See http://web.expasy.org/cgi-bin/blast/blast.pl?action=HTML&sequence=DERTRERLVYEVRQK). Therefore, I can offer a discount off a future purchase if you buy ab4897 now, test it in Mouse and Rat 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 ab4897 in Mouse and Rat. I will then send a discount code. This code must be issued before purchasing ab4897 so please wait for my reply before ordering.

2. Purchase ab4897 either by phone, fax, or online (https://www.abcam.com). No need to mention the code at this stage.

3. Test it in Mouse and Rat.

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: https://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 ab4897 turns out to be unsuitable for Mouse and Rat, 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: https://www.abcam.com/collaborationdiscount.

Thank you for your reply.
I am sorry to confirm it is not possible to provide a blocking peptide on this occasion. I apologise for the inconvenience, I understand this would have been useful in this case.

I hope the other suggestions provided will be helpful and look forward to hearing from you with the outcome. Please do not hesitate to let me know if you have any further concerns regarding the results. I hope we can resolve this case for you as soon as possible.

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from these antibody.

I would like to reassure you that these antibodies are tested and covered by our 6 month guarantee for WB and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a creditnote, free of charge replacement or refund.

As requested, I am forwarding a copy of the general WB testing protocols for these antibodies. Please note that these will be a guideline only and may require some further individual optimization by the end user.

ab4897 Anti-Dnmt3a antibody:
A. Preparation of cell lysates
1. Collect cells (confluent T-25) by trypsinization and spin.
2. Lyze the pellet with 100 ul lysis buffer on ice for 10 min. For 500,000 cells, lyze with 20 ul.
3. Spin at 14,000 rpm (16,000 g) in an Eppendorf microfuge for 10 min at 4‹C.
4. Transfer the supernatant to a new tube and discard the pellet.
5. Determine the protein concentration (Bradford assay, A280, or BCA)
6. Take x ul (= y ug protein) and mix with x ul of 2x sample buffer.
7. Boil for 5 min and cool at RT for 5 min.
8. Flash spin to bring down condensation prior to loading gel.
B. Polyacrylamide gel (14.5 cm ˜ 16.5 cm)
1. Agarose plug:
1% agarose dissolved in 1 ˜ Resolving gel buffer.
2. Resolving gel:
24 ml of a 9% gel
5.4 ml 40% acrylamide/bisacrylamide (29:1 mix)
3 ml 8x Resolving gel buffer
15.6 ml water
12 ul TEMED
60 ul 20% ammonium persulfate
3. Stacking gel: 8 ml
1 ml 40% acrylamide/bisacrylamide (29:1 mix)
2 ml 4 ˜ Stacking gel buffer
5 ml water
8 ul TEMED
21.6 ul 20% ammonium persulfate
C. Preparation of gel
1. Assemble the glass plates and spacers (1.5 mm thick).
2. Pour an agarose plug (1-2 mm).
3. Pour the running gel to about 1 cm below the wells of the comb (˜20 ml).
4. Seal with 1 ml water-saturated 1-butanol. (Can stop here and leave gel as is overnight if you want.)
5. When gel has set, pour off the butanol and rinse with deionized water.
6. Pour the stacking gel (˜5 ml) and insert the comb immediately.
7. When the stacking gel has set, place in gel rig and immerse in buffer.
8. Prior to running the gel, flush the wells out thoroughly with running buffer.
D. Running the gel
1. After flash spinning the samples, load into the wells.
2. Be sure to use markers. We use 15 ul Bio-Rad Kaleidoscope Prestained Standards #161-0324 directly.
3. Run with constant current (35-37 mA with voltage set at >150 V).
4. Usual running time is about 1.3 hr.
E. Using precast gels (Ready Gels from Bio-Rad)
1. Assemble gel in gel rig.
2. Prepare protein samples (10 ug will suffice).
3. Use 5 ul of Kaleidoscope standard.
4. Run at 200 V (constant voltage) for 30 min.
F. Preparation of membrane
1. Cut a piece of PVDF membrane.
2. Wet in methanol on a rocker at RT for 5 mins. Remove methanol and add 1x Transfer buffer until ready to use.
G. Membrane transfer
1. Assemble "sandwich":
2. Prewet the sponges, filter papers (slightly bigger than gel) in 1 ˜ Blotting buffer. Sponge - filter paper - gel - membrane - filter paper - sponge
3. Transfer for 1 hr at 15 volts at 4oC on a stir plate. Bigger proteins might take longer to transfer. For a Mini-Transblot, it's 100 V for 1 hr with the cold pack and prechilled buffer.
4. Immerse membrane in Amido-Black stain 5 mins.
5. Destain 4 ˜ 5 mins with destaining buffer.
6. When finished, immerse membrane in Blocking buffer and block for one hour at room temperature.
H. Antibodies and detection
1. Incubate with primary antibody diluted to 2 ug/ml in total volume of 3 mL in Blocking buffer for one hour at room temperature.
2. Wash 4 ˜ 5 min with 0.05% Tween 20 in TBS.
3. Incubate with secondary antibody diluted 1:10,000 (eg HRP anti-rabbit) in Blocking buffer for 1 hour at room temp.
4. Wash 4 ˜ 5 min with 0.05% Tween 20 in TBS.
5. Detect with Chemiluminescent kit.
I. Stripping blot
1. Rinse blot off with 0.05% Tween 20 in PBS.
2. Put blot into Kapak bag cut to slightly bigger size than blot.
3. Add about 5 to 10 ml Stripping buffer.
4. Remove as much air as possible and seal bag.
5. Immerse into 80‹C water bath and incubate for 20 min.
6. Rinse blot off with 0.05% Tween 20 in PBS.
7. Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween 20.
Buffers for Westerns
Lysis buffer:
0.15 M NaCl
5 mM EDTA, pH 8
1% Triton X100
10 mM Tris-Cl, pH 7.4
Just before using add:
1:1000 5 M DTT
1:1000 100 mM PMSF in isopropanol
1:1000 5 M ƒÃ.aminocaproic acid
2x sample buffer:
130 mM Tris-Cl, pH8.0
20% (v/v) Glycerol
4.6% (w/v) SDS
0.02% Bromophenol blue
2% DTT
8x Resolving gel buffer: 100 ml
0.8 g SDS (add last)
36.3 g Trizma base (= 3 M)
Adjust pH to 8.8 with concentrated HCl
4x Stacking gel buffer: 100 ml
0.4 g SDS (add last)
6.05 g Trizma base (= 0.5 M)
Adjust pH to 6.8
10x Running buffer: 1 L
30.3 g Trizma base (= 0.25 M)
144 g Glycine (= 1.92 M)
10 g SDS (= 1%)--add last


ab119282 Anti-Dnmt3b antibody:
Western blot Protocol (Colorimetric)
Blocking
1. Block membrane by incubating 1 hour at room temperature with shaking in Blocking Solution (5% nonfat dry milk, 0.05% Tween-20 in TBS (50mM Tris, 100mM NaCl, pH 7.6).
Incubation with Primary Antibody
1. Dilute primary antibody at the appropriate dilution in Blocking Solution.
2. Incubate the membrane with diluted primary antibody for overnight at 4℃ with agitation.
3. Remove antibody solution. Wash the membrane 3 times for 10 minutes each time at room temperature in TBST (50mM Tris, 100mM NaCl, 0.05% Tween-20, pH 7.6) with shaking. Note: Increase the concentration of Tween-20 to 0.1% reduces the background and increases the specificity, but it will reduce the sensitivity.
Incubation with Second Antibody
1. Incubate membrane with secondary AP conjugate diluted (according to manufacturer’s instructions) in Blocking Solution for 1 hour at room temperature with shaking.
2. Wash the membrane 3 times for 10 minutes each time at room temperature in TBST (50mM Tris, 100mM NaCl, 0.05% Tween-20, pH 7.6) with shaking.
3. Wash membrane with TBS for 2-5 minutes.
Stain
1. Warm up BCIP/NBT Solution (according to manufacturer’s instructions) to room temperature before use.
2. Apply enough of BCIP/NBT solution to cover the specimen completely. Allow 5-10 minutes, under room temperature, for the color to develop.
3. Monitor the color development until desired intensity is observed.
4. Rinse the specimen gently with distilled water to stop the reaction.
Peptide Competition
Before proceeding Western Immunoblotting, add Blocking Peptide to the diluted primary antibody in a molar ratio of 10:1 (peptide to antibody) and incubate the mixture at 4℃ for overnight or at room temperature for 2 hours.
For ab119282, 50 ug of total protein (HT29 cell lysates) was loaded in each lane on an 10% SDS-Page gel. The antibody was used at a concentration of 1 ug/ml diluted in 1% nonfat dry milk, 0.05% Tween-20 in TBS.

I hope these will be helpful to you. However, if you still have some concerns regarding the results I would be pleased to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you are also able to provide images which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.

Order Details
Antibody code:
Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background
Lot number
Purchase order number
or preferably Abcam order number:
General Information
Antibody storage conditions (temperature/reconstitution etc)
Description of the problem (high background, wrong band size, more bands, no band etc.)
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
Amount of protein loaded

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Detection method (ECL, ECLPlus etc.)
Positive and negative controls used (please specify)
Optimization attempts (problem solving)
How many times have you tried the Western?
Have you run a "No Primary" control?
Yes No
Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?
What steps have you altered?
Additional Notes:
Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

The originator used T47-D cell lysate as a positive control for ab4897 in Western blotting. If you have any more questions, please let us know.

To our knowledge, this antibody has yet to be tested in this application. All tested applications are specified on Abcam product datasheets. If you decide to go ahead and purchase this product, please let us know how you get on and in return we will forward a reward of your choice, typically an Amazon gift voucher.