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Dear technical support team:
This customer has purchased ab3493 (Anti-Dnmt3L antibody) and has conducted the WB several times with mouse sample. The results show high background and non-specific signal; therefore this customer wants to ask for your help to modify her experiment step, could you please offer any suggestion to improve her result?
I attached the image in this letter and her experiment step as follow:
1. Order details:
Batch number: GR24271-3
Abcam product code: ab3493
Antibody storage conditions (temperature/reconstitution etc): -20C
2. Please describe the problem (high background, wrong band size, more bands, no band etc).
High background, many non-specific band, No target band
3. On what material are you testing the antibody in WB?
· Species: mouse
· What’s cell line or tissue: germ cell, ES cells, MEF, 3T3
· Cell extract or Nuclear extract: cell extract
· Purified protein or Recombinant protein: Recombinant protein
3. The lysate
How much protein was loaded: 20 ug
What lysis buffer was used: RIPA
What protease inhibitors were used: Fermentas ProteoBlock
What loading buffer was used: LONZA ProSieve ProTrack
Phosphatase inhibitors: NO
Did you heat the samples: temperature and time: YES, 95C, 3 mins
4. Electrophoresis/Gel conditions/ Transfer conditions
Reducing or non reducing gel: non reducing gel
Gel percentage : 10%
Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, transfer 1hr, 250 mA, pre-stain markers and internal control GAPDH were seen.
5. Blocking conditions
Buffer: 6% milk in PBST, or Millipore BLOK-CH, or 6% BSA
Blocking agent: milk, BSA, serum, what percentage: 6% milk or Millipore BLOK-CH, or 6% BSA, each has been used.
Incubation time: over night
Incubation temperature: 4C
6. Primary Antibody
Reacts against: Dnmt3L
· At what dilution(s) have you tested this antibody:1000x, 500x
· What dilution buffer was used: PBST, Calbiochem SignalBoost™Immunoreaction Enhancer
· Incubation time: 2 hrs
· Incubation temperature: RT
· What washing steps were done: PBST wash 30mins for 5 times
7. Secondary Antibody
Reacts against: anti-rabbit IgG
At what dilution(s) have you tested this antibody: 2000X, 5000X
Incubation time: 1hr, 2hrs
Wash steps: PBST wash 30mins for 5 times
Fluorochrome or enzyme conjugate:HRP
Do you know whether the problems you are experiencing come from the secondary? NO, the secondary antibody has been used successfully for other primary antibody. We can see the non-specific bands.
8. Detection method
ECl, ECl+, other detection method: ECL
9. Did you apply positive and negative controls along with the samples? Please specify.
YES. We used wild type and Dnmt3L knockout ES cells. The target protein expression has been verified by other worked primary antibody and Q-RT-PCR.
10. Optimization attempts
· How many times have you tried the Western? For 5 times.
· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control):
YES. The non-specific bands were not from the secondary antibody. It’s OK seeing non-specific bands. We cannot see the differences of target band between WT and Dnmt3L KO ES cells.
· Do you obtain the same results every time e.g. are background bands always in the same place? Yes
· What steps have you altered?
Primary and secondary incubation time.
The antibody dilution.
Using the Calbiochem signal boost
Using the Millipore Blok-CH
Could you please help this customer to solve the problem?
Thanks for your kindly help.
Asked on Mar 02 2012
Thank you for your enquiry regardingab3493 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer ishaving problems with this antibody.
After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:
1) Immunoprecipitation: As the datasheet indicates, ab3493 worked in WB following IP, but did not work in a straight WB. Please advise your customer to perform an IP firts.
2) Transfection and KO: As teh customer has used transfected cells lines and KO cells, it would be great if you could provide some more details:
2.1 Does the construct contain full sequence or fragment of the target protein?
2.2 Have you used any tagging which may interact with the recognition of the epitope?
2.3 How have you selected the sequence of siRNA? Have you tried more than one SiRNA sequence? Could you check if the sequence corresponds correctly toDnmt3L protein?
2.4 Have you checked the KO with Southern blot application?
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.
Answered on Mar 02 2012