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
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
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)
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.
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.
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.
Choose: Non-specific band Multiple bands No signal or weak signal High background
Purchase order number
or preferably Abcam order number:
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?
Do you obtain the same results every time?
e.g. are the background bands always in the same place?
What steps have you altered?
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.