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Name: Dawinbio Dawinbio
Product code: 30782
Lot number: GR26814-1
Inquiry: Non-specific band I transfected siRNA against PHD3 #1,#2, and #3 . Three days later, siRNA was verified by RT-PCR and western blot with abcam antibody. RT-PCR data showed successful knockdown of PHD3 (see attached figure)but western blot with Ab30782 did not show any reduced PHD3. Recently we got our own custom ab against PHD3 which detected endogenous PHD3 and exactly matched with RT-PCR data.
1) Abcam product code ab
2) Abcam order reference number or product batch number
2-1) storage temperature of antibody
3) Description of the problem
Non specific detection of PHD3
4) Sample preparation:
Type of sample and species(whole cell lysates, fraction, recombinant protein…and Human,
mouse…): whole cell lysate, 1% SDS, TRis buffer, endogenous PHD3, human 786-O cell
Lysis buffer : 1% SDS, Tris buffer
Protease inhibitors:Complete mini
Phosphatase inhibitors : sigma co.
Reducing agent DTT
Product name Anti-PHD3 ab
Cat no Ab30782 Batch no GR26814-1
구매시기 Mar, 2012 구매수량 1
Boiling for ≥5 min? Yes
Protein loaded ug/lane or cells/lane 25 ug
Positive control: siRNA of PHD3 transfected
Negative control : non transfected cell lysate
5) Percentage of gel 10% SDS-PAGE
Type of membrane NC
Protein transfer verified Prestained molecular marker
Blocking agent and concentration 5% non-fat dried milk in TBST
Blocking time One and half hour
Blocking temperature at Room temperature
6) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1:1000
Diluent buffer 1% non-fat dried milk in TBST
Incubation time overnight
Incubation temperature: 4°C
7) Secondary antibody:
Reacts against: rabbit IgG
Concentration or dilution 1:10000
Diluent buffer 5% non-fat dried milk in TBST
Incubation time: one hour
Incubation temperature: room temperature
Fluorochrome or enzyme conjugate:HRP
8) Washing after primary and secondary antibodies:
Number of washes three times, 5 min each
9)Detection method: Thermo scientific, SuperSignal West Pico
10) How many times have you run this staining? two
-Do you obtain the same results every time? yes
-Have you run a "No Primary" control? no
-What steps have you altered to try and optimize the use of this antibody? siRNA , shRNA tried
Document attachment: Attaching images of your blot is strongly recommended and can greatly
speed up our investigation of your problem.
I transfected siRNA against PHD3 #1,#2, and #3 . Three days later, siRNA was verified by
RT-PCR and western blot with abcam antibody.
RT-PCR data showed successful knockdown of PHD3 (see attached figure)but western
blot with Ab30782 did not show any reduced PHD3.
Recently we got our own custom ab against PHD3 which detected endogenous PHD3 and exactly matched with RT-PCR data.
Asked on May 04 2012
Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hearthe customer has had difficulty obtaining satisfactory results from this antibody.
The details you have kindlysent will provide us with vital information for our monitoring of product quality. I would like to reassure you that this antibody is tested and covered by our guarantee for WB and human samples.
I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.
I am pleased to offer you a free of charge replacement or credit note in compensation.
Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.
Answered on May 04 2012