Anti-LAMP1 antibody [H4A3] - Lysosome Marker (ab25630)

WB with mouse small intestine mucosa
sees bands at 55 and 70 kDa
Ab from other company also shows bands at 55 and 70, but also 120 kDa
Ab: 1/200 o/n
block: 5% milk
2nd: HRP, works well
suggested Hela lysate as positive control as blocking peptide is not available

ANSWER:

Thank you for contacting us.

I have arranged for the shipment of the Hela cell lysate ab29545 on order number xxx.
Please let me know what results you are obtaining and if this helps to interprete your results with the mouse samples.

I look forward to hear back from you and assist you further.

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ab25630: WB with rat liver
sees band at 25 kDa
transfer is OK, detects other proteins in the blot
Ab: 1/1000 1-2 h RT
block: overnight 4 degree
secondary: OK
antibody from other company to same protein gives same results
suggestions: WB conditions might not yet be optimal, try blocking for only 1-2 h RT, antibody incubation: overnight 4 degree
ab25631: rat is unsuitable
ab2773: WB with rat liver, sees band at 25 kDa
suggestions: WB conditions might not yet be optimal, try blocking for only 1-2 h RT, antibody incubation: overnight 4 degree

ANSWER:

Thank you for contacting us.
I am sorry to hear about the problems you have encountered.

The bands you are seeing at 25 kDa could be the light chain of endogenous IgG.

As we discussed over the phone, please try the following suggestions as it seems the WB conditions for these 2 antibodies (ab25630 and ab2773) are not yet optimal:

1) try blocking for only 1-2 h RT
2) try the primary antibody incubation for overnight 4 degree

As for ab25631, this has been shown to not react with rat samples and would probably not give satisfactory results.However, if you have access to a human sample, you could try the antibody on that.

Please let me know if the tips were helping to improve your results.

I hope this information is helpful to you, and I look forward to hear back.

human WT fibroblast cell line, Lamp2 k/o cell line
sees expected band in WT cells, but sees no band in lamp2 k/o cells
is Ab specific to lamp1?
tried other Ab from other company and gets expected results

Ab: 1/500, 1/300, 1/1000 overnight
blockig: 55 milk
secondary:OK

ANSWER:

Thank you for contacting us.

Your credit note ID is xxx.

I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

The credit note ID is for your reference only and does not automatically guarantee the credit.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

Many thanks for your reply. I would like to ask you if you could send me a vial of the other Anti- LAMP1 antibody [H4A3] (ab119127) please which you had offered to me as a replacement.

I really appreciate your help.
Kind regards,

ANSWER:

Thank you for confirming these details and for your cooperation.

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 a free of charge replacement of alternative LAMP 1 antibody with the order number 1019233.

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 letme know.

I wish you the best of luck with your research.

Please find attached the questionaire you sent to me regarding to my enquiry about the LAMP1 antibody (ab25630).

Please let me know if there is anything else you need to know and what your decision about a replacement antibody is.
Many thanks for your help!

Kind regards,

Order Details
Antibody code: LAMP1 antibody (ab25630)
Lot number:GR64381-1

General Information Antibody storage conditions (temperature/reconstitution etc)
The antibody was delivered on 12th January 2012 in storage buffer consisting of 100mM Borate buffered saline, pH 8.2 without preservatives. As recommended the antibody was aliquoted and stored at -20 C. Description of the problem (high background, low signal, non-specific satining etc.)

There is no signal at all on my embryonic sections. I have tried 3 different dilutions 1:50, 1:75 and 1:100 and the staining looks as the negative control.

Sample (Species/Tissue/Cell Type/Cell Line etc.)
Paraffin-embedded embryonic mouse sections containing heart, lung, skin, spinal cord and bone tissue were used.
Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
The tissue sections were dewaxed and rehydrated in 100%, 90%, 80%, 70%, and 50% ethanol. Non-specific binding was blocked in TBS/Tween20 (0.5%) and 10% rabbit serum for 1 h at RT. The primary mouse anti-LAMP1 antibody was incubated in a humidified chamber at 4 °C over night. After washing 3 times the sections were incubated with a secondary Alexa555 rabbit anti-mouse antibody for 2h at room temperature. The sections were washed 3 times and nuclei were stained with DAPI. The sections were mounted in Prolong antifade (Invitrogen).

Antigen retrieval (Enzymatic method, Heat mediated technique etc.) ForAntigen Demasking slides were immersed in citrate buffer (pH 6.0) and Antigen Retrieval was performed in a pressure cooker (Biocare Medical, UK) for 2 min at 125oC followed by 10 min at 80oC

Permeabilization step
The permeabilization step was done together with the blocking.

Blocking conditions (Buffer/time period, Blocking agent etc.)
Non-specific binding was blocked in TBS/Tween20 (0.5%) and 10% rabbit serum for 1 h at RT.

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) LAMP1, Abcam, tested at 3 different dilutions (1:50, 1:75, 1:100)in in TBS/Tween20 (0.3%) and 3% rabbit serum

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) rabbit anti-mouse Alexa555 IgG (1:200 in 1M TBS, pH 7.4, Invitrogen, A-21427)

Detection method
Fluorescence-Microscopy, Confocal Microscopy Positive and negative controls used (please specify)

negative control: 1 section was incubated in TBS omitting the primary antibody
positive control : no

Optimization attempts (problem solving)
I have tried to use lower dilutions of the antibody up to 1:50.

How many times have you tried the IHC?
I have the IHC 3 times.
Have you run a No Primary control? Yes

Do you obtain the same results every time?
Yes What steps have you altered? I have changed the dilutions for the primary antibody. I haven't changed anything else as the protocol is optimised and works very well for all my other antibodies.As I want to combine this antibody with 2 other antibodies I wouldn't like to change that protocol at all.

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 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 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 trying the secondary antibody with another appropriate primary antibody to asses if this is working.

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