Anti-Thrombospondin antibody [A6.1] (ab1823)

I’m attaching the blot with TSP1 that I ran today. There are 4 pieces there – The first 2 are the TSP1 (1:200) and negative control (the small one-no TSP1) blocked with 5% milk and 5% serum for an hour. The tonsil control was ran in the lane next to the ladder (lane 2). As you can see there is no band at 140kda. There is a lighter band lower (by the splotch) that is similar to the TSP1 in the odyssey blot to the right.

The next 2 blots were ran with odyssey blocking buffer (TSP1 1:200) and the negative control. On this blot, the Tonsil is located in the 8th lane (lane #1 is the ladder). In this lane, you will notice very light staining of TSP1 at around 140kda and another band around near the bottom with a bubble in the gel.

Protein concentrations of brain lysates ranged from 20-75 ug/well (20, 40, 60, 75; at time points where TSPs have gene upregulation). Placement was randomized (which is why the Tonsil is in 2 different locations) to account for the potential of Tonsil lysate spill-over when loading the well . The ladder in gel 2 is longer than 1, so take that into account when comparing bands.

From these results we conclude

1. that you must use the odyssey buffer to visualize any kind of TSP1 band.

2. That even with 40 ug of tonsil (the most we can load in our wells) there is minimal staining (and 2 bands).

3. That in rat brain lysate (cortex) of 75ug tissue, TSP1 staining is not evident, even though we’ve visualized TSP1 staining in IH using this Ab.

Unless you have alternative suggestions, we would like a refund for the TSP1 as our needs for western blot have not been met as advertised (and I don’t have enough Ab to try another go).

I greatly appreciate your advice and support; however for the application we require this antibody for, I feel we are at a dead end.

ANSWER:

I am sorry that the antibody did not prove to be successful for your needs and as requested I have gone ahead and processed a refund for the cost of ab1823. Your credit note number is ********.

Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

Please let me know if there is anything else I can help you with.

Couple of questions!

1. How many ug of protein do you recommend running in a well to pick up TSP1 in the tonsil? 40ug (which is the most I can run) is what I am preparing to try.

2. Our protocol is to heat our proteins to 70C for 10 minutes. Your protocol says boil for 5 minutes but does not specify the boiling point of your storage buffer. Specifics would be handier than trial by error here, if you have them. Otherwise I’ll heat to bp of water (100C) for 5 minutes.

Thanks for letting me harass you!

ANSWER:

I am glad you enjoyed the conference.

For boiling, I would heat the water 100C and then add the sample for 5 minutes. As for how much protein to load, I would go ahead and load 40ug of lysate, as we want to know if the antibodyis working correctly and it’s better to have to high a signal in this case than a faint band.

Please let me know how it goes and if there is anything else I can help you with.

We will be leaving for a conference this weekend and won’t return until the beginning of March. I think the lag time will fine. Please send the Tonsil lysate when it comes back in stock.



Thank you for your terrific service!

ANSWER:

Thank you for your reply.

I have processed the tonsil lysate order (there will be no actual charge though) and it is set to ship as soon as it come back into stock. You should hopefully receive it at the end of February or the beginning of March.

I hope you enjoy the conference and that the tonsil lysate proves to be successful. If there is anything else I can help you with, please let me know.

That information is correct. I’ve attached the invoice in case you have any other questions.

I’ve ordered the Blocking buffer and will compare with 5% milk.

We concluded that this may be a secondary issue, which is why we ordered a different secondary. I will adjust the concentration according to package standards in the future.

If I can get a nice blot with the brain lysate and positive control, would you like me to send you a picture?

ANSWER:

Thank you for your reply.

The tonsil lysate that i was going to send you is currently out of stock, we have a lead time of 1-2weeks for it to come back into stock. We have some tumor tonsil lysate, but I am uncertain as to whether this will work for you or not.

I am sorry about this, please let me know how you would like to proceed, in terms as to whether you still would like the tonsil lysate, of if you want to test out your new secondary and block first.

I look forward to your reply.

I am purchasing the Li-cor block at your recommendation. Out of curiosity though, what is your normal recommended block for TSP1 in rat brain? We have several li-cor secondary Abs and don’t have any issue with the block that we use. Due to the heavy background on the first blot, I didn’t think I was over-blocking, however our normal protocol is 60 minutes.

My negative condition 75ug of protein loaded, Blocked, No Primary Ab, same Secondary Ab. (same conditions without a primary Ab added to the block for the overnight incubation).

Thank you so much for responding so quickly. I’m quite enthusiastic to get this to work!! I hope that with the sample of tonsil we can figure a working protocol!

Please let me know a recommended amount of protein to add for a control. That would be very helpful!

ANSWER:

Thank you for your reply.

I think I have found your order but I just want to make sure as the invoice number you gave is not in our system (the order I found was paid for by credit card).


For normal HRP-tagged secondary antibodies we would recommend using either 5% milk or 5%BSA as a blocking reagent. The reason I was concerned about blocking in your case is because as you clarified in your last email, your negative control, was when you applied the secondary antibody only to the membrane and you got the exact same staining pattern as seen with your primary + secondary condition. This makes me think that the problems due to the initial blot you did was due to the non-specific binding of the secondary antibody to the membrane.

It could be that you also used to high a concentration of secondary antibody the first time (1/3000) and that may have contributed to the high background that you were seeing.

Once you have confirmed that the original order information, I can send the tonsil lysate as a positive control.

I look forward to your reply.