Overview

  • Product name
    Anti-PDGF A antibody
  • Description
    Rabbit polyclonal to PDGF A
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ELISA, WBmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Human PDGF A (N terminal) conjugated to keyhole limpet haemocyanin.

  • Positive control
    • HL60 cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab38562 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ELISA 1/1000.
WB 1/100 - 1/500. Detects a band of approximately 24 kDa (predicted molecular weight: 24 kDa).

Images

  • Anti-PDGF A antibody (ab38562) at 1/100 dilution + HL60 cell lysate

    Predicted band size: 24 kDa
    Observed band size: 24 kDa
    Additional bands at: 36 kDa, 40 kDa, 75 kDa. We are unsure as to the identity of these extra bands.

References

This product has been referenced in:
  • Cofer ZC  et al. Methylation Microarray Studies Highlight PDGFA Expression as a Factor in Biliary Atresia. PLoS One 11:e0151521 (2016). WB . Read more (PubMed: 27010479) »
See 1 Publication for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Question

Thank you for you kindly help in this case.
This customer had used the new Lot you kindly provided.
Unfortunately, it still gave no target signal and high background. Would you please help this customer deal with issue?
Here is the detail:
1. Order details:

Batch number: Lot GR52858-1

Abcam product code: ab38562

ORDER number: 1027098

Antibody storage conditions (temperature/reconstitution etc) -20℃
2. Please describe the problem (high background, wrong band size, more bands, no band etc).
No band at 24KD
3. On what material are you testing the antibody in WB?

Species: human

What’s cell line or tissue: HL-60, C2C12, HEPM

Cell extract or Nuclear extract: Cell extract

Purified protein or Recombinant protein: total cell lysate
3. The lysate

How much protein was loaded: 30ug

What lysis buffer was used: RIPA (50mM Tris-HCl pH 8.0; 150 mM NaCl; 1% NP40; 0.5% Sodium Deoxycholate 0.1%SDS; 5mM EDTA )

What protease inhibitors were used: Roche protease inhibitor

What loading buffer was used: 4X protein loading dye (40% glycerol, 240mM Tris-HCl pH6.8, 8% SDS, 20% β-ME, 0.1% Bromophenol blue)

Phosphatase inhibitors :no

Did you heat the samples: temperature and time: yes, boiling 7 mins
4. Electrophoresis/Gel conditions/ Transfer conditions

Reducing or non reducing gel: reducing gel

Reducing agent: SDS

Gel percentage : 10%

Transfer conditions: (Type of membrane, Protein transfer verified): NC paper
5. Blocking conditions

Buffer: TBS buffer

Blocking agent: milk, BSA, serum, what percentage:5% milk

Incubation time:1 hours

Incubation temperature: room temperature
6. Primary Antibody

Species: Rabbit

Reacts against: PDGF

At what dilution(s) have you tested this antibody: 1:100

What dilution buffer was used: 5% milk in TBS

Incubation time: over night

Incubation temperature: 4℃

What washing steps were done: 1 time/15mins,total 1hours
7. Secondary Antibody

Species:

Reacts against: Rabbit

At what dilution(s) have you tested this antibody: 1:8000

Incubation time: 1 hours

Wash steps: 1 time/15mins,total 1hours

Fluorochrome or enzyme conjugate: enzyme

Do you know whether the problems you are experiencing come from the secondary? Secondary antibody is ok!!
8. Detection method
ECl, ECl+, other detection method: ECL
9. Did you apply positive and negative controls along with the samples? Please specify.
HL-60, C2C12
10. Optimization attempts

How many times have you tried the Western? 3 times

Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control):

Do you obtain the same results every time e.g. are background bands always in the same place? yes

What steps have you altered?
Thank you very much.
Best regards

Read More
Answer

Thank you for contacting us. I have checked the image which is not very nice. The black dots explains, the buffers should be filtered. Could you try filtering all the buffers?
- Wash the primary and secondary antibody with TBST for 10 minutes 1 time and for 5 minutes 3X

Have you used the same protocol successfully before?

Looking forward to hearing from you soon.

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1130116 (ab54518).

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Answer

Thank you for your email.

The explanation of different band sizes was for PDGF AA protein. It is the protein that customer is interested in, so I am sorry the experiments has to be determined by end user. We unfortunately cannot do experiment in our lab to prove the band detected is protein PDGF-AA; it is not unknown protein.

We apologize for customer's unexpected results so we are happy to replace their antibody; alternatively a credit note can also be given. Could you letus know how the customer would like to proceed?

Thank you for your cooperation. I will be looking forward to hearing from you soon.

Read More

Answer

Thank you for your email.

The PDGF-AA is a homodimer of PDGF-A protein (http://www.uniprot.org/uniprot/P04085). The PDGF-A protein further gets cleaved, amino acid 87-211. Due to these post translational modifications the observed band size would be different than the predicted band size of 24kDa which actually corresponds to unprocessed protein. Secondly the protein is glycosylated at 134th amino acid which also adds up molecular weight.

As this protein exist as homo-dimeric and heterodimeric forms so the observed band size will be different than predicted. Technically, this antibody should not detect the homodimer or hetyerodimer becuase of reduced or denatured conditions however the high molecular weight may indicate the protein is under reduced. so further test may be needed to check the identity of band at 38 kDa.

I am sorry we do not have any further trouble shooting instructions. I hope the instructions give will be helpful.

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Answer

Thanks you for your email. I am sorry for contacting you late because I was in touch with lab. My colleague has confirmed that they have observed a band of 24kDa with HL60 cell line lysates when characterized this ab. So I am sorry I am unable to find the reason why this antibody is giving band at 38kDa with your lysates. The Ponceau looks OK. I can offer you a vial from different lot and hoping that the new vial could deliver the best results you are 0065pectiong. Let me know how you would like to proceed.

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Question

LOT NUMBER 940685 ORDER NUMBER 958784 DESCRIPTION OF THE PROBLEM No signal at 24KD SAMPLE •Species: human •What’s cell line or tissue: HL-60, C2C12, HEPM •Cell extract or Nuclear extract: Cell extract •Purified protein or Recombinant protein: total cell lysate PRIMARY ANTIBODY •Species: Rabbit •Reacts against: PDGF •At what dilution(s) have you tested this antibody: 1:200 •What dilution buffer was used: 5% milk in TBS •Incubation time: over night •Incubation temperature: 4℃ •What washing steps were done: 1 time /15mins,total 1hour DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED HL-60, C2C12 ANTIBODY STORAGE CONDITIONS -20℃ SAMPLE PREPARATION •What lysis buffer was used: RIPA (50mM Tris-HCl pH 8.0 150 mM NaCl 1% NP40 0.5% Sodium Deoxycholate 0.1%SDS 5mM EDTA ) •What protease inhibitors were used: Roche protease inhibitor •What loading buffer was used: 4X protein loading dye (40% glycerol, 240mM Tris-HCl pH6.8, 8% SDS, 20% β-ME, 0.1% Bromophenol blue) •Phosphatase inhibitors :no •Did you heat the samples: temperature and time: yes, boiling 7 mins AMOUNT OF PROTEIN LOADED 30ug ELECTROPHORESIS/GEL CONDITIONS •Reducing or non reducing gel: reducing gel •Reducing agent: SDS •Gel percentage : 10% TRANSFER AND BLOCKING CONDITIONS •Transfer conditions: (Type of membrane, Protein transfer verified): NC paper Blocking conditions •Buffer: TBS buffer •Blocking agent: milk, BSA, serum, what percentage:5% milk •Incubation time:1 hours •Incubation temperature: room temperature SECONDARY ANTIBODY •Species: •Reacts against: Rabbit •At what dilution(s) have you tested this antibody: 1:8000 •Incubation time: 1 hours •Wash steps: 1 time/15mins,total 1hours •Fluorochrome or enzyme conjugate: enzyme •Do you know whether the problems you are experiencing come from the secondary? Secondary antibody is ok!! HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

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Answer

Thank you for your enquiry regarding ab38562 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. Additionally, thank you for supplying an image, as this has been very beneficial in understanding your concerns. The protocol looks fine to me however I would like to ask few questions which would help me identify the source of the problem; - Have you used the same protocol successfully before? - It may well be that the protein wasn't transferred properly. Have you had a chance to do Ponceau staining? I will look forward to hearing from you soon.

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