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Dear technical support team:
This customer has purchase ab89436 (Anti-RED1 antibody) and has conducted the wb with human sample. The results show the expression was doesn’t decrease of knowdown sample; 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 image in this letter and her experiment step as follow:
1. Order details:
Batch number: gr37579-2
Abcamproduct code: ab89436
Antibody storage conditions (temperature/reconstitution etc) 20℃
2. Please describe the problem (high background, wrong band size, more bands, no band etc).
The expression was doesn't decrease of knowdown sample
3. On what material are you testing the antibody in WB?
· Species: human
· What’s cell line or tissue: HeLa cell
· Cell extract or Nuclear extract: Cell extract
· Purified protein or Recombinant protein:
3. The lysate
How much protein was loaded:50 ug
What lysis buffer was used: WCE
What protease inhibitors were used: PI (Roche)
What loading buffer was used:
Phosphatase inhibitors :NaF, Na4VO3, souium pyrophosphate, beta-glycerolphosphate
Did you heat the samples: temperature and time:100℃, 10min
4. Electrophoresis/Gel conditions/ Transfer conditions
Reducing or non reducing gel:Reducing gel
Reducing agent: DTT
Gel percentage :8%
Transfer conditions: (Type of membrane, Protein transfer verified): PVDF,
5. Blocking conditions
Blocking agent: milk, BSA,serum, what percentage: 5% non-fat milk
Incubation time: 1 hr
Incubation temperature: RT
6. Primary Antibody
Reacts against: human
At what dilution(s) have you tested this antibody: 1/1000
What dilution buffer was used: 5% non-fat milk in 1X TBST
Incubation time: O/N
Incubation temperature: 4℃
What washing steps were done: 5 times, 1X TBST
7. Secondary Antibody
Species: goat-anti-mouse IgG
At what dilution(s) have you tested this antibody: 1/5000
Incubation time: 1 hr
Wash steps: 5 times, 1X TBST
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. Other antibody and siRNA-treated cells
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):
Do you obtain the same results every time e.g. are background bands always in the same place? Yes
What steps have you altered? no
Could you please help this customer to solve the problem?
Thanks for your kindly help
Asked on Apr 10 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 this antibody.
The details you have kindly sent will provide us with vital information for our monitoring of product quality
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 are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial. I have been in contact with the originator and theyhave kindly offered toretest this antibody. I can provide you with further information as soon as this has been done.
I apologize for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation.
In addition, I can suggest you may like to consider the following suggestions as a check for future experiments:
1. For siRNA knockouts, we would suggest it is beneficial to always consider including an endogenous negative control to help assess how well the knock out and antibody procedure are working.
2. We have the following document regarding siRNA experiments on our website which I hope may be helpful to you:
Thank you for your cooperation and patience. I look forward to hearing from you with details of how you would like to proceed.
Answered on Apr 10 2012