Anti-KLF4 antibody (ab26648)

Hello,
I am looking for an antibody against human KLF4 that works in EMSA.
I looked through your website but none of the antibodies have been validated for this technique. Could you help me select the right one, perhaps one that has shown to work in CHIP as this will ensure that the epitope recognised by the ab is not in the DNA binding domain.
Kind Regards,

ANSWER:

Thank you for enquiry and your interest in our products.

Currently, testing our antibodies in EMSA is not part of the standard QC process. Most of our products are characterized regularly in WB, IHC, ICC, ELISA, Flow cytometry, ChIP.

Currently, we have 8 antibodies against human KLF4. It may be worth considering an antibody which recognizes the native form of the protein so that protein-DNA or protein-RNA interactions can be studied in EMSA. Currently, none of these antibodies have been tested specifically in ChIP, however those which work in IHC/ELISA may be suitable for this application:

ab10846,

ab26648,

ab72543,

ab75486,

ab118961,

I could offer you a discount code for testing in EMSA if you select one of these antibodies and happy to co-operate with us. The Terms and Conditions of this offer can be found at: http://www.abcam.com/collaborationdiscount.

Let me know if you wish to proceed with the discount code and confirm the product code number of your interest.

I hope this helps and if I can assist further, please do not hesitate to contact me.

LOT NUMBER gr29399-1 ORDER NUMBER 986370 DESCRIPTION OF THE PROBLEM Multiple bands SAMPLE Species: mouse and human * What’s cell line or tissue: mouse ESC, human ESC, human HepaG2 * Cell extract or nuclear extract: cell extract PRIMARY ANTIBODY Primary Antibody:anti-KLF4 Species:rabbit polyclonal, react with human & mouse Reacts against: *At what dilution(s) have you tested this antibody: 1:500 & 1:1000 *What dilution buffer was used: 5% milk in PBST *Incubation time: O/N *Incubation temperature: 4 ℃ *What washing steps were done: 3 times * 10 min/each 6 times * 10 min/each DETECTION METHOD chemofluorescence ANTIBODY STORAGE CONDITIONS -20 ℃ SAMPLE PREPARATION What lysis buffer was used: 1% triton X-100 What protease inhibitors were used: What loading buffer was used: Phosphatase inhibitors: Na3Vo4 Did you heat the samples: temperature and time: 100 ℃, 10min AMOUNT OF PROTEIN LOADED 30 μg ELECTROPHORESIS/GEL CONDITIONS Gel percentage : 10 % TRANSFER AND BLOCKING CONDITIONS Buffer:PBST Blocking agent: milk, BSA, serum, what percentage:5% milk Incubation time:1 hr & 2 hr Incubation temperature: RT SECONDARY ANTIBODY Species:rabbit Reacts against: At what dilution(s) have you tested this antibody: 1:5000 Incubation time:1 hr Wash steps:3 times with 10min each, in PBST HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? concentration of primary antibody and blocking buffer

ANSWER:

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 provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimize the results from ab26648. I would also appreciate if you can confirm some further details:  

1. For lysis, when for 1% Triton, does this mean a lysis buffer such as RIPA containing all the salts and other components as well as the Triton?  

2. Please confirm if sample buffer containing SDS and mercaptoethanol added before heating to reduce and denature?

3. I can recommend to check the cells not over confluent when they are lysed if this has not already been done, to ensure they are healthy.  We would recommend to ensure they are about 70-80% confluent and to check the viability which should be over 95%.

4. Please confirm if a loading control has been included in order to assess quality of the samples.

5. I suggest to consider trying BSA rather than milk to block. Changing blocking agent can sometimes improve the results.

6. Please provide further information regarding the secondary antibody. Which supplier and product code? Is the current vial of secondary antibody working well with other primary antibodies?

In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund. I would like to reassure you that ab26648 is covered by our guarantee in WB and in human samples. 

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.