Anti-Dnmt3b antibody - ChIP Grade (ab2851)

LOT NUMBER XXXX ORDER NUMBER XXXX DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE RAT NORMAL EMBRYO , total protein PRIMARY ANTIBODY Concentration or dilution 1:100-1:400 Diluent buffer 1:10 of blocking Incubation time O/N Incubation temperature 4°C Washing: Buffer Used PBST Number of washes 6 washes WITH 5 MINS CHANGE DETECTION METHOD ECL advance POSITIVE AND NEGATIVE CONTROLS USED Positive control NA Negative control NA ANTIBODY STORAGE CONDITIONS -20degC SAMPLE PREPARATION Lysis buffer RIPA Protease inhibitors COMPLETE PROTEASE Phosphatase inhibitors - Reducing agent DTT, MERCAPTO Boiling for ≥5 min? YES AMOUNT OF PROTEIN LOADED Protein loaded ug/lane or cells/lane 40ug ELECTROPHORESIS/GEL CONDITIONS Reducing or Non Reducing gel REDUCING Percentage of gel 10% Volts applied 100V Time applied 60-70 mins TRANSFER AND BLOCKING CONDITIONS Type of membrane NC Protein transfer verified YES Blocking agent and concentration 5% blocking provided in ECL advane kit( from amersham) Blocking time 1 hr Blocking temperature RT SECONDARY ANTIBODY Species Swine Reacts against Rabbit Concentration or dilution 1:10,000 Diluent buffer 1:10 of blocking Incubation time 1 hr Incubation temperature: RT Fluorochrome or enzyme conjugate HRP Washing: Buffer Used PBST Number of washes 10 washes WITH 5 MINS CHANGE HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Changing protein Concentration, blocking %, primary antibody dilution from 1:100 to 1: 400, secondary antibody dilution, washing time etc, ADDITIONAL NOTES The another antibody received with above mentioned (i.e. anti-SP3) worked fine with the same sample and same protocol indicating that there is no problem with the sample or protocol and secondary Ab.

ANSWER:

Thank you for contacting us and taking the time to send the questionnaire. We really appreciate all the details provided as well as the images sent, that help us to better understand the problem. All of our products are covered by our Abpromise guarantee; which ensures that you can trust our products, and they should work in the tested species and applications stated on the datasheet, or we will offer a replacement, credit, or refund, if reported within 6 months of purchase. I would really like to help you solve the problem and obtain good results from this antibody, and for that I am very pleased to give you some protocol tips. In the case these suggestions don’t improve the results, please let me know, and I will be happy to send a replacement or a credit note. -In order to enrich the signal, prepare nuclear lysates to increase the protein levels in the sample. -During sample preparation, it is crucial to use protease inhibitors as well as phosphatase inhibitors to avoid sample degradation. -I would suggest trying a different blocking agent. Have you tried 5%BSA for 1-2 hours at RT? -Decreasing the secondary concentration may also help to improve the signal. -To avoid smearing of the gel, using fresh prepared buffers is crucial. - You may run a positive control to assess how well the antibody is working. This protein is highly expressed in JEG-3 lysates. I hope these tips help, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again, and I’ll be more than happy to help you further.

My apologies. I was not being clear. I need a good positive control target for anti-DNMT1 ChIP.

ANSWER:

A brief literature search revealed that PMID: 16357870 (Vire E et al Nature 2006 439:871) has used U2OS cells and primers specific for the hsMYT1 promoter. The primers are described in PMID: 15231737 (Kirmizis A et al Genes and Dev 2004 18:1592).

I hope this information will be useful,

Our customer confused about the molecular weight you mentioned. They found that all referneces they searched showed that the molecular weight of this protein is ~130 kDa. The attachment file is the reference which were published in Vol. 439 at Feb 16 last year. This data is very important for our customer to carry on their research. We would be appreciated if you could help them elucidate this case, thank you!

ANSWER:

Thank you for getting back in touch with me. To clarify.

A predicted molecular weight of 97KDa for Dnmt3b is derived from SwissProt reference Q9UBC3. This is purely based on the protein sequence and a prediction. ab2851 detects a band of approximately 130 kDa which correlates with the Dnmt3b reference that you have provided me with.

I can tell you that this antibody also detects recombinant DNNMT3b1 at ~130KDa and recombinant DNMT3b3 at ~100 kDa; this may explain the additional bands.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

I apologize for a delay in my response, but I didn't have a chance to reply before. I am sending the photo of the DNMT3B blot, I really don't know what happened with the prevous attachment. So, these are the bands that I am seeing when I use your antibody, upper one (which I believe is specific) is around let say 90 Kd and the lower (probably unspecific one) is around 80 Kda. And to answer your questions... Incubation with the primary antibody was overnight. And, no, I didn't try blocking with BSA with this Ab. I usually try that when I have very weak signal. In this case I have had pretty strong signal with very strong nonspecific band. So, I am sure background would be much more pronounced with BSA than with nonfat dry milk.I didn't find it reasonable to try when I already had very strong nonspecific band. Please let me know what you think about it.

ANSWER:

I have been advised by the source of ab2851 that it would be most important to run a positive control. In the lab recombinant DNMT3b has been used, however it is reported that DNMT3b is highly expressed in P19 (murine EC) cells when compared with human cells such as HeLa or HCT116. I would therefore recommend to run P19 cell lysate.

It would be worth trying to block the membrane to see if the BSA alters the banding pattern. It would also be worth running a negative control (no primary antibody) to determine if the non-specific banding is being caused by interaction with the secondary antibody.

An interesting point was raised by my colleagues: splice variants: It is known that there are at least 5 different splice variants for DNMT3b, and it is also believed that the protein is post-translationally modified. Therefore it is not unexpected to see some size heterogeneity.

Finally, I may be able to add to the catalogue some neutralizing peptide to determine which bands are specific if you are interested in trying this.

I hope the advice detailed above is useful, please do not hesitate to contact me if you have further questions,

BATCH NUMBER 216131 ORDER NUMBER 184828

DESCRIPTION OF THE PROBLEM There are (at least) 2 bands, around 95 and 85 kDa. According to what you stated (130 kDa) both bands appear to be of wrong size. However, since expected MW of the protein is around 100 kDa, most likely the upper band is specific and the lower band is not specific (in support of that the upper band does and the lower band doesn't disappear after DNMT3b siRNA treatment). It's been impossible to get rid of this lower (nonspecific) band since it's too strong, even stronger than the upper (specific) one.

SAMPLE NT-2 cell nuclear extract (NT-2 cells- human cell line - neuronal precursor cells)

PRIMARY ANTIBODY Abcam, ab2851, rabbit polyclonal, 1:500 and 1:1,000 dilutions tried, 3x 10 min-washes with PbS.

DETECTION METHOD ECL plus

POSITIVE AND NEGATIVE CONTROLS USED We don't have appropriate positive control for this antibody (please include one if you have it). But, I have control (non-treated) sample and sample treated with siRNA for DNMT3b - please see the attachment.

ANTIBODY STORAGE CONDITIONS aliquoted and stored at -20 (as recommended).

SAMPLE PREPARATION PIERCE kit is used for nuclear extract preparation. protease inhibitors are added in each step during preparation of the sample. After addition of 5xloading buffer, samples are boiled for 10 min. and run on a gel.

AMOUNT OF PROTEIN LOADED 15 and 7.5 microg (20, 10 and 5 microg of protein have also been tried).

ELECTROPHORESIS/GEL CONDITIONS 8% Tris-Glycine gel (Invitrogen) - 1.5h, 125V, 35mA.

TRANSFER AND BLOCKING CONDITIONS transfer buffer: Tris/Glycine/20% Methanol, 4h-transfer (25V, 125 mA); blocking buffer -PbS/0.1% Tween/5% nonfat dry milk (1h-blocking).

SECONDARY ANTIBODY Amersham, ECLTM Anti-rabbit IgG, Horseradish Peroxidase linked, 1:2,000 dilution, 2h-incubation, 3x 10 min-washes with PbS.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 8 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Tried with different concentration of the antibody (1:500 and 1:1,000), different amount of protein loaded (5-20 microg), different concentration of the gel (8%, 10-20%, 4-20%) , different transfer buffer (w/ or w/o methanol), different transfer time (2h, 4h and o/n)...

ANSWER:

Thank you for contacting us for technical support.

Unfortunately we cannot find the attachment you refer to and I would be very grateful if you could send it again, so I can investigate your results in better details.

Can you please also clarify how long you have incubated the primary antibody for and if you have tried blocking in 5% BSA?

Thank you very much for letting me know this information and sending the image of your results, I look forward to hearing from you to help you better,