Question (55319) | Anti-KIF4A/KIF4 antibody (ab3815)

Go to datasheet (ab3815)

Question

Product code: 3815
Lot number: GR86868-1

Inquiry: Antibody code: ab3815
Batch number: GR86868-1
General Information:
Antibody storage conditions (temperature/reconstitution etc) -20deg
Description of the problem (high background, low signal, non-specific staining etc.) non-specific staining, no relevant signal
Sample (Species/Tissue/Cell Type/Cell Line etc.) HELA cells
Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
Tried both Methanol/Acetone/ or Paraformaldehyde
Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Permeabilization step Aceton for Methanol, Triton for formaldehyde
Blocking conditions (Buffer/time period, Blocking agent etc.) 5% BSA, 1h
Primary Antibody (Diluent/Dilution/Incubation time, Wash step) 1h RT 1:100, 3 washes PBST
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time,
Wash step) Alexa anti goat 594, 1:200, 1h RT
Detection method Observer. Z1 Microscope (Zeiss)
Positive and negative controls used (please specify) negative control – no primary Optimization attempts (problem solving):
How many times have you tried the IHC? 2 times
Have you run a "No Primary" control? yes Do you obtain the same results every time? yes
What steps have you altered?
Additional Notes
Document Attachment: images of the results may be very helpful. If you wish to add it to the complaint form, please attach them to the reply e-mail.

Antibody code: ab3815 Batch number: GR86868-1
Antibody storage conditions (temperature/reconstitution etc) -20
Description of the problem (high background, wrong band size, more bands, no band etc.) no relevant band, many non-specific bands
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) HELA cell extract, 293-T cell extract, HELA and 293-T synchronized with Nocodazole for 12h lysates
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) RIPA buffer + protease inhibitors (merk)
Amount of protein loaded 30microg
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) reducing gel 10%
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) 1h 20min semi-dry transfer, TGB +20% MetOH transfer buffer, blocking with 5% milk
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) 1:100 in 1% BSA solution, 1h RT or ON 4deg, 6 washes with PBST
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Anti goat 1:5000 (Jackson), 1% BSA, 1h RT, 6 washes with PBST
Detection method (ECL, ECLPlus etc.) ECL Positive and negative controls used (please specify) OPTIMIZATION ATTEMPTS (PROBLEM SOLVING)
How many times have you tried the Western? 3 times Have you run a "No Primary" control?
Do you obtain the same results every time? e.g. are the background bands always in the same place? yes
What steps have you altered? ON incubation with ab, cell syncronization Additional Notes

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.

I would like to reassure you that ab3815 is tested and covered by our 6 month guarantee for use in WB, ICC-IF and inhuman samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimise the results. I would also appreciate if youare able to confirm some further details:

ICC-IF

1. Has the quality of the sample been assessed, for example what was the% confluency before staining, and was a spare sample checked for viability using trypan blue count before staining?

2. Fixation: To prevent over fixation, whichever method is used, I can suggest this should be for 10 minutes only if this has not already been tried. PFA should be 4% only.

3. Please provide further information regarding the permeabilization. I would suggest to try 0.2% triton, 10 minutes only.

4. I can recommend to consider reducing the concentration to 1:500 to reduce the background signal.

5. I would appreciate if you are able to provide an image which would help us to assess the results.

WB

1. I can suggest to try reducingthe concentration to 1:500 to reduce the background signal. Incubate overnight 4oC.

2. Could you confirm if the quality of sample been assessed using a loading control?

3. Have the samples reduced and denatured?

4. I would appreciate if you are able to provide an image which would help us to assess the results.

General:

1. Please confirm the order number and date of purchase.

2. Is the current vial of secondary antibody working well with other primary antibodies?

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.

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