Anti-GAPDH antibody [mAbcam 9484] - Loading Control (ab9484)

Abcam product code ab9485

2) Abcam order reference number or product batch number GR64234-1
3) Description of the problem: Tried dilutions 1:2500, 1: 1250 and 1:625 (blocking either with 5% Milk or 5% BSA). Even at 1:625 the signal is still too weak and unspecific bands appear.
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): Crude brain lysate
Lysis buffer: 1x Laemmli buffer
Protease inhibitors: none
Phosphatase inhibitors: none
Reducing agent: beta-Mercaptoethanol contained in Laemmli Buffer
Boiling for ≥5 min? yes
Protein loaded ug/lane or cells/lane not known
Positive control: brain lysate itself
Negative control: none
5) Percentage of gel: 4-20% TGX gradient Gel
Type of membrane: PVDF
Protein transfer verified: yes
Blocking agent and concentration (5% milk or 5% BSA in TBST)
Blocking time: 1h
Blocking temperature: RT
6) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution: 1:2500, 1: 1250, 1:625
Diluent buffer: Milk or BSA
Incubation time: o/n
Incubation temperature: 4°C
7) Secondary antibody: Cy3 goat anti-rabbit IgG (H+L)
Species: goat
Reacts against: rabbit
Concentration or dilution: 1:2500 or 1: 1250
Diluent buffer: Milk or BSA
Incubation time: 1h
Incubation temperature: RT
Fluorochrome or enzyme conjugate: Cy3
8) Washing after primary and secondary antibodies:
Buffer: TBST
Number of washes: 5x 5 min
9)Detection method: Imaging system with CCD camera
10) How many times have you run this staining? 4 times
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? Primary antibody concentration and blocking medium, dilution of 2ndary AB
Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

ANSWER:

Vielen Dank für Ihre Antwort und für diese weiteren Informationen.


In der Tat sieht es so aus, als ob der Antikörper unter diesen Bedingungen funktioniert haben müsste. Zwar bekommen Sie ein Signal, aber es ist in der Tat viel zu schwach bei dieser hohen Konzentration (auch wenn man schön den Unterschied zwischen Milch und BSA sehen kann).


Ich weiß, dass Sie viel Zeit für diese Experimente verwendet haben und möchte Ihnen daher einen Ersatz oder einen Gutschein als Ersatz anbieten (vorausgesetzt, das Produkt wurde in den letzten 180 Tagen gekauft). Dafür benötige ich jedoch ihre ursprüngliche Bestellnummer oder einen Kontakt in Ihrer Einkaufsabteilung und das Datum der Bestellung. Würden Sie mir bitte zusammen mit diesen Informationen mitteilen, welche Alternative Sie bevorzugen?

Vielen Dank für Ihre Kooperation und Hilfsbereitschaft. Ich freue mich bald wieder von Ihnen zu hören.

In customers original contact email with us they did mention that antibodies were purchased last year, they just did not have time to report the technical issue until now. They were in the midst of running multiple projects last year as well. Please let me know if credit cannot be issued and I will advise the customer.
Kind regards,

ANSWER:

Thank you for your response.
In future, we would appreciate if the problem is reported as soon as possible and to make sure that Australian customers are aware of our Abpromise.
This is to let you know that I have just contacted and asked our Account Department to raise a credit note for you - for the cost of one vial of ab9484. For your information, the internal reference note for this credit is CN20759. You can use this credit in the near future for any of the products which are in the catalogue.
I hope this helps and if I can assist further, please do not hesitate to contact me.

AB9484, Lot# GR27709, our PO# PO-10654, order was placed on 07/06/11.
Hope this helps.
Kind regards,

ANSWER:

Thank you for confirming the order number.
I regret to inform you that this order (894340) was placed last year (2011) and as Abpromise expired.
Would you be so kind to explain why the problem was reported more than 10 months after purchase (27/04/2012)?
As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
If you need any further assistance in the future, please do not hesitate to contact me.

Product code: 9484
Lot number: GR27709

Inquiry: Please have a look at the technicial inquiry form attached for the details of the problem.

ANSWER:

Thank you for providing the Western blot images.
In order to raise the credit note, I need to find the original order in our system. Could you please provide the Abcam Order Number and the date of purchase?
It would be very much appreciated if batch and order confirmation could be given in the first contact.
Many thanks and I look forward to hearing from you soon.

Please see customers reply below:

Overloading wasnt the case. I can't remember if I attached the images or not but you can see it that in the exact same replica of the gel stained with HA ANTIBODY is fine.

Thanks for your help. We will be processing a credit for the customer as requested.

Kind regards,

ANSWER:

Thank you for your response.
I am afraid the image has not been attached and it would be much appreciated if you could send it with your next e-mail. This would certainly help us to find the source of the problem during our monthly analysis.
Would you be so kind to do that and I will raise the credit note accordingly.
I look forward to hearing from you soon.