GSK3 beta (GSK3B) ELISA Kit (ab123454)

Below are my comments to your response. Please let me know if you have anymore suggestions.
If the sample is giving negative results after background subtraction, it means that the GSK3B is not detectable in that sample.
When you look at the other file I sent where the concentration of protein was 0.5 mg/mL I was able to get positive readings for my samples. But then when I tested several different protein concentrations the 0.5 mg/mL sample was negative. I believe that the sample was negative because my blank was so high.
It seems that the only positive numbers in the excel file were sample loaded at 2 and 2.5mg/mL. This seems a very high protein concentration. What protein assay did you use? We recommend using the BCA assay since the detergent that comes with the kit does not interfere with this protein assay.
The protein assay that I used was the Detergent Compatible Protein Assay from Bio-rad.
My recommendation would be to re-test the protein concentration of the sample. How many cells did you use to make the sample extract? How much volume of detergent did you add?
At most for one sample I am using about 8 million cells. After I count the cells I determine how much of the extraction buffer to add.
The day to day variability in the blank can be caused by fluctuations in assay temperature or reagent mixing. So long as the standard readings are linear, your assay results will be reliable.
If the blank is varying day to day this is a problem because I am subtracting it from my sample results and if it is high one day and low another day then this is causing my results to be positive on one day and negative on another day.

ANSWER:

Thank you for your reply.

When using the recommendedab63193 as a standard curve,if the background increases, the raw numbers of the calibration curve will also increase (but everything should be relatively proportional). The interpolation of the sample from the standard protein should not change from day to day more than 10%.

Also, in the protocol it is emphasized that the samples should be loaded as soon as they are thawed.Our lab hasfound that GSK3B is extremely labile and leaving the samples thawed for even 1 hour before loading will change the levels in comparison to loading immediately after thaw (this is specified in the protocol). Extracts should not be frozen and I recommend to use a fresh extract every time the assay is performed.

I hope this helps to improve your results. If not, and you have purchased this kit in the past six months, I will be happy to offer you a free of charge replacement, credit or refund. Please let me know your original order number and how you would like to proceed and I will be happy to assist you further.

I am using ab123454 and the blank absorbance values keep changing from day to day. I seem to be getting negative sample readings. How much protein do you recommend loading?

ANSWER:

Thank you for contacting us.

If the sample is giving negative results after background subtraction, it means that the GSK3B is not detectable in that sample. It seems that the only positive numbers in the excel file were sample loaded at 2 and 2.5mg/mL. This seems a very high protein concentration. What protein assay did you use? We recommendusing theBCA assay since the detergent that comes with the kit does not interfere with this protein assay. My recommendation would be to re-test the protein concentration of the sample. How many cells did you use to make the sample extract? How much volume of detergent did you add?

The day to day variability in the blank can be caused by fluctuations in assay temperature or reagent mixing. So long as the standard readings are linear, your assay results will be reliable.

human pancreatic cancer cell lysate
expects to see drop in GSK3B amount after treatment
standard curve looks fine
at 0.5 mg/ml lysate: values at 2-4 ng/ml and difference between control and treated samples seen
at 2.5 mg/ml lysate: values around 20 ng/ml and BUT no difference between control and treated samples seen anymore. Could the well be saturated at this conc?

ANSWER:

Thank you for contacting us.

I heard back from the lab with the following advice:

We would suggest youto load in the working range of the assay for the pancreatic cell extract. As an example we show in the protocol the working range for two types of extract (HeLa and H4IIE). As you will see in the protocol, the range will change depending on the cell type and the species.
The lack of difference at 2.5mg/mL loading is probably due to oversaturation of the well.

We would suggest totest the kit using a titration series of untreated pancreatic cell extract. This titration would be very useful to understand the linearity of the assay on that particular cell line you are usingand to be able to load at the right concentration for accurate measurement of GSK3B protein quantity.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Is the concnetraion of 2x10/7cells per ml correct in the protocol?

ANSWER:

Thank you for calling Abcam.
I talked to the lab about the protocol for ab123454 and they have confirmed that the concentration on part 8.1.3 of the protocol is correct and that is the concentration of cells that is required.
Please let me know if there is anything else I can help you with.