Anti-Glucose 6 Phosphate Dehydrogenase antibody (ab993)

DESCRIPTION OF THE PROBLEM We obtain two bands one next to the other around the expected size. Depending on the tissue, one or the another band is more abundant. I don?t know whether there is a reference published with this antibody, specially in mouse tissues to see how the western looks

SAMPLE Tissue homogenates

DETECTION METHOD ECL Plus

POSITIVE AND NEGATIVE CONTROLS USED It is a housekeeping gene, then we have signal in every tissue

ANTIBODY STORAGE CONDITIONS -20?C

SAMPLE PREPARATION Buffer+protease inhibitors

AMOUNT OF PROTEIN LOADED 20 ?g

SECONDARY ANTIBODY Goat anti-rabbit peroxidase conjugated

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

Thank you for your enquiry regarding a reference for ab993.

The following reference has used this antibody: Jain M et al. Increased myocardial dysfunction after ischemia-reperfusion in mice lacking glucose-6-phosphate dehydrogenase. Circulation 109:898-903 (2004). PubMed: 14757696

The method used in that paper was the following:

"Total G6PD protein levels were determined by standard Western blot. Frozen hearts were homogenized in Triton X-100 lysis buffer (Cell Signaling) with protease inhibitors (2 umol/L leupeptin, 1 mmol/L PMSF). The lysate was centrifuged at 16 000g for 15 minutes at 4°C. The supernatant was collected, and protein concentration was measured according to the method of Lowry et al.21 Samples were run on 12% Tris-glycine precast gels and transferred to polyvinylidene difluoride membranes. Equal protein loading was confirmed by Ponceau staining. After blocking in 5% nonfat milk, polyvinylidene difluoride membranes were probed with rabbit anti-rat G6PD IgG followed by horseradish peroxidase–conjugated anti-rabbit secondary antibody. Protein levels were detected by chemiluminescence and quantified by densitometry."

From the figure on that paper I was unable to see two bands in those mice heart samples, however on the image on our datasheet there is a faint band under the strong band which may be a variant of G6PD. Another possibility is that the band is a breakdown product and you may need to consider higher levels of protease inhibitors in your lysis buffer and making sure the samples are on ice at all times.

I hope this information helps, please do not hesitate to contact us again if you require further assistance,

I have the following enquiry from a customer, I looked on the data sheet and did not see any information on the expected size: Thank you for your help. Question: I have multiple bands after probing with G6PD on a Western blot.What is the expected size?

ANSWER:

On the datasheet for ab993, there are two blots shown with a strong band at approx. 65-70 kDa (antibody was used at 1:5000). If the customer has any more questions, please contact us again.

I would like to know how to dilute this sample for use in Western Blot. Thank you

ANSWER:

Try this antibody at 1:1000 and adjust accordingly from the results obtained.