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DESCRIPTION OF THE PROBLEM Non-specific band
SAMPLE mouse lung whole cell lyasate
PRIMARY ANTIBODY Concentration or dilution : 1:500 Diluent buffer : 0.1% TBS-Tween Incubation time : overnight Incubation temperature: 4℃ Washing Buffer 0.1% TBS-Tween Number of washes 5 X 5 mins
DETECTION METHOD using LAS-1000 (Fuji)
POSITIVE AND NEGATIVE CONTROLS USED negative control : normal mouse lung cell
ANTIBODY STORAGE CONDITIONS -20 ℃
SAMPLE PREPARATION Lysis buffer : novagen phophosafe reagent Protease inhibitors: added Reducing agent: added (2-ME and DTT) Boiling for ≥5 min? yes/no : no
AMOUNT OF PROTEIN LOADED Protein loaded ug/lane or cells/lane : 60ug/lane
ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE Gel
TRANSFER AND BLOCKING CONDITIONS Type of membrane : PVDF (pore size :0.45 um) Protein transfer verified : verify using pre-stained protein marker Blocking agent and concentration : 5% non-fat milk for non-phosphorylated type and 5% BSA for phosphorylated type protein Blocking time: 1 hr Blocking temperature : RT
SECONDARY ANTIBODY Species: goat Isotype: IgG Reacts against: rabbit IgG Concentration or dilution : 1:2000 Diluent buffer : 0.1% TBS-Tween Incubation time : 1.5 hrs Incubation temperature: RT Fluorochrome or enzyme conjugate: HRP Washing Buffer 0.1% TBS-Tween Number of washes 5 X 5 mins
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? I altered Ab concentration, incubation time and temperature
ADDITIONAL NOTES I would like to confirm which bands on blotted membranes are my target protein. Size (molecular weight) of band is different from that of datasheet |
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ANSWER: |
Thank you for contacting us. The mouse 5-lipoxygenase, like the human protein is 77.9 kDa. You would therefore expect to see a band at around this size. However, this size can vary considerably depending on the cell lysate produced, how the gel is performed and the buffers used. You appear to have specific bands from both antibodies appearing at around 60kDa. With a non-specific band with the Phospho-5-Lipoxygenase at around 45 kDa.
In your protocol you mention that you used both DTT and b-mercaptoethanol. This is usually not necessary and it should be sufficient to use either DTT (final concentration of 20 mM) or b-mercaptoethanol (final concentration 5%). Additionally, unless you are attempting to run a native gel I would suggest heating the samples for 5 minutes at 95-100°C to allow the proteins to denature. You do not mention which buffers were used but the ones we typically recommend can be found here:
http://www.abcam.com/index.html?pageconfig=resource&rid=11375
Additionally in order to check if the antibody is specifically recognising the phosphorylated form of the protein you could de-phosphorylate the protein as described here:
http://www.abcam.com/index.html?pageconfig=resource&rid=11407
I would also strongly recommend carrying out a no primary control to ascertain if this is contributing to the specificity. I hope this information has been of help to you, if you remain unsatisfied with the performance of this antibody please let me know. |
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