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Dear technical support team:
This customer has purchased ab38621 (Anti-BNIP3 antibody) and has conducted the wb with mouse sample. The results show no bands, therefore, she wants to ask for your help to modify her experiment step, could you please offer any suggestion to help her?
I also attached her data in this letter and her experiment step as follow:
1. Order details:
Batch number: XXXXX
Abcam product code: ab38621
Antibody storage conditions (temperature/reconstitution etc) -20℃
2. Please describe the problem (high background, wrong band size, more bands, no band etc).
3. On what material are you testing the antibody in WB?
· Species: Mouse
· What’s cell line or tissue：liver
· Cell extract or Nuclear extract: cell extract
· Purified protein or Recombinant protein: Purified protein
3. The lysate
How much protein was loaded: 88 ug
What lysis buffer was used: EBC lysis buffer
What protease inhibitors were used: protease inhibitor (sigma)
What loading buffer was used: 4X loading dye
Phosphatase inhibitors ：no
Did you heat the samples: temperature and time:100℃ 10 min
4. Electrophoresis/Gel conditions/ Transfer conditions
Reducing or non reducing gel:
Gel percentage : 13％
Transfer conditions: (Type of membrane, Protein transfer verified): PVDF 100V 90 min
5. Blocking conditions
Blocking agent: milk, BSA, serum, what percentage: 5％ milk
Incubation time:1 hr ℃
Incubation temperature: RT
6. Primary Antibody
Reacts against: human and mouse
· At what dilution(s) have you tested this antibody:1:200
· What dilution buffer was used: 5％ milk in TBST
· Incubation time: overnight
· Incubation temperature: 4℃
· What washing steps were done: four time , 10 min/each
7. Secondary Antibody
At what dilution(s) have you tested this antibody: 1:5000
Incubation time: 1 hr RT
Wash steps: 15 min once, 10 min once, 5 min five times
Fluorochrome or enzyme conjugate:HRP
Do you know whether the problems you are experiencing come from the secondary? NO
8. Detection method
ECl, ECl+, other detection method: ECl
9. Did you apply positive and negative controls along with the samples? Please specify. NO
10. Optimization attempts
· How many times have you tried the Western? 2 times
· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no
· Do you obtain the same results every time e.g. are background bands always in the same place? yes
· What steps have you altered? Protein loading
Could you please help this customer to solve the problem?
Thanks for your kindly help
Asked on Apr 24 2012
Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from our products.
Reviewing this case, I would like to offer some suggestions to help optimise the results from ab8399.
-I would recommend the use of a positive control, as it is a good indicator of the antibody performance. Mouse brain tissue or Ramos cell lysate are recommended.
-BNIP3L is a phosphorilated protein. Milk is not recommended as a blocking agent when phospho-proteins, as the casein could break the phosphorilated positions. Instead, I would recommend using 5% BSA for 1 hour at RT, which in addition is a milder blocking agent and can help visualize the bands of interest.
- Increase the protein rate in your samples, using a mitochondrial lysate instead of a total cell lysate. RIPA buffer could be a good choice to do it.
-Increase the antibody concentration. Try 1/50 or 1/100 in an attempt to improve the results.
-For the washing process I would recommend using TBST, and perform 4 washes of 10 minutes each.
I hope this suggestions help improve the results from the antibody. Please do not hesitate to contact us for further assistance.
Answered on Apr 24 2012