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1) Abcam product code ab76024 2) Abcam order reference number or product batch number lot:GR1737-3 3) Description of the problem The antibody is not detecting any catalase in my samples that have previously shown a large amount of catalase by mass spectrometry.See attached pptx file which in yellow highlights all peptides detected by mass spectrometry on same sample. Catalase is in my sample based on many spectra and peptides identified for this protein. 4) Sample preparation: Type of sample: Proximal convoluted cells isolated from rat kidneys and organelle(mitochondria, peroxisomes, & etc) isolated by sucrose gradient Lysis buffer: RIPA Protease inhibitors: PMSF Phosphatase inhibitors; NONE Reducing agent: BME Boiling for ≥5 min? Yes and no, I have tired both ways Protein loaded ug/lane or cells/lane Positive control: Whole cell lysate from Proximal convoluted cells isolated from rat kidneys which little is detected. Negative control 5) Percentage of gel: 10% Type of membrane: PVDF Protein transfer verified: YES Blocking agent and concentration: Thermo Non-protein blocking buffer Blocking time: 2 hours Blocking temperature: RT 6) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution Diluent buffer: Blocking buffer with 0.1% Tween Incubation time: O/N Incubation temperature:4 degrees 7) Secondary antibody: Species: mouse Reacts against: Rabbit Concentration or dilution: 1:10,000 Diluent buffer Blocking buffer with 0.1% Tween & 0.01% SDS Incubation time: 30 minutes Incubation temperature: RT Fluorochrome or enzyme conjugate:Fluorochrome 8) Washing after primary and secondary antibodies: Buffer; TBS 0.1% Tween Number of washes 3X 9)Detection method 10) How many times have you run this staining? All the time nothing new in our lab. Do you obtain the same results every time? What steps have you altered to try and optimize the use of this antibody? The following dilutions of primary tired: 1/10,000, 1/5,000, 1/2,000, 1/1,000 all O/N 4degrees See attached file and let me know if you need more details.
Asked on Sep 09 2011
Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this product. I have been discussing this issue with some colleagues in the Lab. Regrettably, I have been informed that ab76024 is no longer recommended for use in rat samples based on internal test results. I have just amended the on-line datasheet to reflect this. I appreciate the time you have spent on these experiments in the laboratory and it is disappointing the results have not been successful. I am very sorry for this discrepancy and can certainly offer you either an immediate refund or a credit note which you can use in the future. Please do let me know how you wish to proceed with your enquiry.
Answered on Sep 09 2011