Anti-IGFBP1 antibody (ab4242)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Non-specific band

SAMPLE rat retina extract

PRIMARY ANTIBODY rabbit polyclonal to IGFBP1, ABCAM, ab4242

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED NO control

ANTIBODY STORAGE CONDITIONS -20C

SAMPLE PREPARATION Protease inhibitors

AMOUNT OF PROTEIN LOADED 20ug

ELECTROPHORESIS/GEL CONDITIONS 10% reducing Gel

TRANSFER AND BLOCKING CONDITIONS transfer 1hr, good. blocking 4C overnight with 5%milk

SECONDARY ANTIBODY Goat anti-rabbit HRP 2nd antibody,promega 1:2500, 5%milk/TBST, for 1hr.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No I need some help with explanation for the data based on your product rabbit polyclonal to IGFBP1(ab4242) which claim the predicted molecular weight is 32KDa, But My blot shows a clear band around 90 kDa with no other visible band at 32kDa and other size. I am at a loss to give an explanation, could you help me with that? thanks.

ANSWER:

I have received the following additional information from the laboratory, which I hope will help you.

IGFBP1 is definitely localized in rat hepatoma cells, but it may not be located in rat retina (only in diabetic and hypoxic rats). See references below:

"IGFBP-1 was not detected within the rat eye." Investigative Ophthalmology & Visual Science, Vol 37, 1459-1468 (1996) Investigative Ophthalmology and Visual Science 46:2708-2715 (2005)"

Please also find below a summary of the Western Blot protocol.

Western Blot Protocol 1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a H-4-II-E cell conditioned media sample and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water. 2. Block the blotted nitrocellulose in freshly prepared PBS containing 3% nonfat dry milk (PBS-MLK) for 20 minutes at 20-25°C with constant agitation. 3. Incubate the nitrocellulose with 1ug/ml of IGFBP-1, diluted in freshly prepared PBS-MLK overnight with agitation at 4°C. 4. Wash the nitrocellulose twice with water. 5. Incubate the nitrocellulose in the secondary reagent of choice (a goat anti-rabbit HRP conjugated IgG, 1:3000 dilution was used) in PBS-MLK for 1.5 hours at room temperature with agitation. 6. Wash the nitrocellulose with water twice. 7. Wash the nitrocellulose in PBS-0.05% Tween 20 for 3-5 minutes. 8. Rinse the nitrocellulose in 4-5 changes of water. 9. Use detection method of choice (enhanced chemiluminescence was used).

I noticed that this was tested on 10X concentrated tissue culture medium. How was the medium concentrated? What sample type was used to test this antibody in mouse? Can this antibody also be used on cell lysates?

ANSWER:

Thank you for your enquiry.

I received the following information from the originator of the antibody:

Working with a rat H-4-II-E hepatoma cell culture supernant, the antibody was purified using Protein A chromatography; then concentrated using Amicon ultrafiltration (YM-30) for storage. For quality controls, the media was diluted 1:10 and run in WB.

Unfortunately, I have no information regarding how mouse IGFBP1 was QC'd.

Regarding detecting IGFBP1 in cell lysates, I would certainly assume that would be possible. While the protein is secreted, as seen in cell culture, obviously the protein is manufactured within the cells.

I hope this information helps. Please do not hesitate to contact us if you need anything further.