Question (56088) | Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody [MAPK-YT] (ab50011)

Go to datasheet (ab50011)

Question

Order Details
Antibody code: ab50011
Lot number:GR121-8
Purchase order number
or preferably Abcam order number: Purchase order number is ####
General Information
Antibody storage conditions (temperature/reconstitution etc)
Stored at 40C
Description of the problem (high background, low signal, non-specific
satining etc.)
This batch gave no nuclear staining as expected, when we worked up the
Ab using GR121-7. All staining was cytoplasmic with a small percentage
of membranous.
Sample (Species/Tissue/Cell Type/Cell Line etc.) Human colorectal
tumour tissue
Fixation of sample
(Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
Formalin fixed, and paraffin embedded.
Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Vector Antigen unmasking solution, 2 minutes in a pressure cooker
Permeabilization step
Blocking conditions (Buffer/time period, Blocking agent etc.) 15
minutes in hydrogen peroxide, and 10 minute is 1x casein block
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation
time, Wash step)
Ab50011 GR121-8, 1 in 50 dilution, diluted in Zymed Antibody diluents
containing 1:50 dilution of casein. Incubated 16 hours at 4 degrees C.
Washed off with TBS.
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation
time, Wash step)
DAKO envision plus mouse secondary HRP-polymer, for 30 minutes. Again
washed off with TBS.
Detection method
DAB, followed by counterstaining with Hematoxylin and lithium carbonate.
Positive and negative controls used (please specify)
Human kidney sections used.
Optimization attempts (problem solving)
How many times have you tried the IHC?
We optimised lot number GR121-7, over several runs, changing dilution
factor, retrieval method, secondary Ab type etc, but when GR121-8 was
run using the identical conditions used to optimise GR121-7, we saw NO
nuclear staining as expected, and previously demonstrated. We have
repeated the GR121-8 run and saw exactly the same staining pattern as
seen previously, where NO nuclear staining was detected.
Have you run a "No Primary" control?
Yes
Do you obtain the same results every time?
Yes
What steps have you altered? NO steps were altered between runs using
GR121-7 and GR121-8, so we expected to have obtained identical
staining patterns.
Additional Notes
We would appreciate if you are also able to provide and image which
would help us to assess the results
We have attached 2 images, one of GR121-7 and GR121-8. Identical
sections were stained with both antibodies, and you can quite clearly
see the difference between the 2 images.

Answer

Thank you for taking the time to complete our questionnaire and provide the images.

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 very few tips to provide on this occasion to help improve the results. I can suggestthat regrettably youmayhavereceived a bad vial.

I can recommend you may like to consider trying the following options which may help provide more consistent results between batches:

1. I can recommend to try serum to block rather than casein. Changing blocking agents can sometimes help to improve results.

2. I can suggest to wash in PBS containing 0.2% Tween. The Tweenwill help to keep the cells permeabilised, and the antibody solubilised.

3. I can recommend toaliquot and store at -20oC or -80oC as directed on the datasheet. This will help to ensure the antibody remains stable and that we can continue to provide our guarantee.

Alternatively, or if these tips do not work, I 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.

Sign up