Anti-Rab4 antibody - Early Endosome Marker (ab13252)

Thank you for your recent reply. I have attahed the blot as a pdf, I hope this is readable. I have also filled in the details below:


Order Details
Antibody code: ab 78547

Problem
Choose: weak signal

Lot number: 874838

Purchase order number
or preferably Abcam order number: Sent as a replacement to ab13252



General Information
Antibody storage conditions (temperature/reconstitution etc)

Aliquots at 4C and -20C

Description of the problem (high background, wrong band size, more bands, no band etc.)
Very weak signal, more bands

Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)

Whole cell lysate

Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)

RIPA buffer


Amount of protein loaded
25-50ug

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
17.5% SDS-PAGE gel

Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)

Transfer 1hr, blocking 1hr milk

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Used at recommended dilution 1:600 overnight. Washed 3x 5min PBS+0.1% Tween

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Anti-rabbit HRP dilution 1:15,000 1hr incubation. Washed 3x10min PBS+0.1% Tween

Detection method (ECL, ECLPlus etc.)
ECL

Positive and negative controls used (please specify)
Positive control: large vol protein from whole cell lysates

Negative control: not relevant


Optimization attempts (problem solving)
How many times have you tried the Western?
At least 5x


Have you run a "No Primary" control?
No

Do you obtain the same results every time?
Yes

e.g. are the background bands always in the same place?


What steps have you altered?
Increased SDS-PAGE gel %, increased incubation with primary duration

Additional Notes:

ANSWER:

Thank you for your response.

My colleague is out of office this week and she has asked me to look after her customers.

There seems to be a faint band at 15 kDa and another stronger one at 60 kDa in the Western blot image you have sent to us.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

TOMM20 is localised to mitochondrion outer membrane. In order to be able to increase the intensity of the lower band, it may be worth making mitochondrial preparation and increasing the final working concentration of this antibody (i.e. 1/500 or 1/300).

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Thank you for your recent reply. I have attahed the blot as a pdf, I hope this is readable. I have also filled in the details below:

Order Details
Antibody code: ab 78547

Problem
Choose: weak signal



Lot number: 874838

Purchase order number
or preferably Abcam order number: Sent as a replacement to ab13252



General Information
Antibody storage conditions (temperature/reconstitution etc)

Aliquots at 4C and -20C

Description of the problem (high background, wrong band size, more bands, no band etc.)
Very weak signal, more bands

Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)

Whole cell lysate

Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)

RIPA buffer


Amount of protein loaded
25-50ug

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
17.5% SDS-PAGE gel

Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)

Transfer 1hr, blocking 1hr milk

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Used at recommended dilution 1:600 overnight. Washed 3x 5min PBS+0.1% Tween

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Anti-rabbit HRP dilution 1:15,000 1hr incubation. Washed 3x10min PBS+0.1% Tween

Detection method (ECL, ECLPlus etc.)
ECL

Positive and negative controls used (please specify)
Positive control: large vol protein from whole cell lysates

Negative control: not relevant


Optimization attempts (problem solving)
How many times have you tried the Western?
At least 5x


Have you run a "No Primary" control?
No

Do you obtain the same results every time?
Yes

e.g. are the background bands always in the same place?


What steps have you altered?
Increased SDS-PAGE gel %, increased incubation with primary duration

Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

ANSWER:

Thank you for your response.

My colleague Kate is out of office this week and she has asked me to look after her colleagues.

I understand that this vial was a free of charge replacement. Could you please summarize if the problem with ab78547 is similar to the original antibody (ab13252) CCE3506002?

This product has been tested for WB application on HepG2 and 1 µg/ml gave a distinctive band. Since the sample types (V2 and 293T cells) are different I would suggest testing higher concentration.

TOMM20 is localised to mitochondrion outer membrane. Have you tried using enriching TOMM20 and separating mitochondrial membranes rather than whole cell lysates?

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Thank you for you reply, and for being able to offer a replacement antibody. Please can I request anti-tomm 20, ab78547, rabbit polyclonal.

Kind regards,

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued an alternativefree of charge replacement of ab78547:

Order number: 1052618

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Thank you for your recent reply to my query regarding antibody ab13252 against rab 4

Answers to the questionsgiven below


2. How has the antibody been stored?

At 4C, with aliquots at -20C

3. Could you confirm how have the samples been prepared? I can recommend to lyse in RIPA lysis buffer to ensure a suitable protien preparation. Then add sample buffer such as lamaeli, including SDS and mercaptoethanol, to heat to reduce and denature.

RIPA has been used to lyse cells. SDS-Page loading buffer was added to samples, which were then boiled at 95C for 2mins then loaded directly onto gels

4. We recommend to load 20 - 30 ug of total protein per lane of the gel to ensure there is enough protein to detect. Althought the GAPDH has provided good bands, this protein is highly expressed. therefore, if there is not so much Rab4 in the cells, this may not be detected unless more protein is loaded.

I have repeated gels with 25ng protein/lane with no improvement in signal unless antibody concentration is also raised

5. I can suggest to try a higher concentration of antibody, such as 1:500. This will help to increase the signal. Incubate overnight at 4oC.

The attached blot shows antibody concentration of 1:200 overnight, yet the intensity of the specific band is not at all comparable to the datasheet.

6. Could you confirm if the current vial of secondary antibody isworking well with other primary antibodies?

Yes, our anti-rabbit HRP has been successfully used with other blots

As the results we have obtained have not been comparable to the datasheet, is it possible to request a replacement for this antibody?

Regards,

ANSWER:

Thank you for taking the time to complete our questionnaire. The details you have kindly provided 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 succesful. 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 recieved a bad vial.

I apologise for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Hello

We purchased an anti-rab4antibody (ab13252) from you a couple of weeks ago (data sheet printed on 23/1), and are having issues getting good western blots using this. The samples used are a murine norovirus infection of RAW cells at 0hrs through to 24hrs, and non-infected 293T cells are used as a control.

The protocol I have used is below.

- Load 10ug sample onto SDS-PAGE gel and run at 200V

- Transfer to PVDF membrane using semi-dry apparatus

- Block with 5% milk for 1hr

- Probe with antibody 1:1000 dilution in 5% milk overnight

- Wash 3x 5mins PBST

- Incubate with secondary anti-rabbit HRP antibody for 1hr

- Wash 3x 10mins PBST

- Use ECL and exposure times from 30s - 5mins

We have also tried using 5%BSA instead of milk but this has not made any improvement.

Probing the same blot with GAPDH using the same protoc0l has confirmed that there is protein present in equal concentrations in every lane.

Any technical suggestions gratefully received.

ANSWER:

Thank you for taking the time to provide this information 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 ab13252 is tested and covered by our 6 month guarantee for use in WB and mouse samples. In the event that a product is not functioning in the applications and species 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 from ab13252. I would also appreciate if you can confirm some further details:

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


2. How has the antibody been stored?


3. Could you confirm how have the samples been prepared? I can recommend to lyse in RIPA lysis buffer to ensure a suitable protien preparation. Then add sample buffer such as lamaeli, including SDS and mercaptoethanol, to heat to reduce and denature.


4. We recommend to load 20 - 30 ug of total protein per lane of the gel to ensure there is enough protein to detect. Althought the GAPDH has provided good bands, this protein is highly expressed. therefore, if there is not so much Rab4 in the cells, this may not be detected unless more protein is loaded.


5. I can suggest to try a higher concentration of antibody, such as 1:500. This will help to increase the signal. Incubate overnight at 4oC.


6. Could you confirm if the current vial of secondary antibody isworking 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 information and details of how you would like to proceed.